Lipopeptides containing an interferon-γ fragment, and uses thereof in pharmaceutical compositions

ABSTRACT

The invention concerns any lipopeptide characterized in that it comprises: a peptide part comprising the peptide sequence consisting of about 30 to about 50 of the last contiguous amino acids of the interferon-γ (IFN-γ) C-terminal end of mammals, whereof, if required, the last 3 to 20 amino acids have been suppressed; and one or several lipophilic parts comprising C4-C20 chain of carbon atoms, saturated or unsaturated, linear or branched, or a steroid group. The invention also concerns any lipopeptide such as defined above containing one or several CD8, and/or CD4, and/or B epitopes. The invention further concerns medicines or vaccines containing any polypeptide such as defined above.

This application is 371 of PCT/FR 99/00259 filed Feb. 5, 1999.

The present invention relates to lipopeptides containing an interferon-γfragment, as well as to their use in particular as medicinal products inthe context of treating or preventing pathologies against whichinterferon-γ is liable to have activity by means of at least one of itsbiological effects, or as an immunity adjuvant which can be used in avaccine composition for stimulating, orienting or re-orienting thebalance of the immune response with respect to any antigen, inparticular by promoting the establishment of a type-1 immune responserelative to a type-2 response with respect to this antigen.

Cytokines, which are cell immunity components, are classified in twogroups: type-1 and type-2 cytokines characterized, respectively, byIL-2, IL-12, interferon-gamma (IFN-γ), IL-4 and IL-5. These two groupsregulate and control themselves mutually. They are closely linked to theinduction and regulation of the immune response.

The expression “type-1 immune response” means the induction of a type-1cytokine profile.

The expression “type-2 immune response” means the induction of a type-2cytokine profile.

On account of its pleiotropic activities, IFN-γ plays a fundamental rolein establishing the immune balance. Specifically, this cytokine isinvolved in various immune defence processes, in particular againstviruses, bacteria and protozoan parasites. Furthermore, it inhibitstype-2 cytokines and promotes the establishment of a type-1 responsewhich is quite often required to combat certain tumours, and to combatviral and parasitic infections.

Consequently, a therapeutic strategy involving this cytokine appearsattractive, but comes up against certain difficulties. Thesedifficulties are the consequences of the short lifetime of IFN-γ,imposing repeated injections of large doses, this often being associatedwith side effects due to the broad spectrum of activity of the molecule.

The production of recombinant human IFN-γ and its use in human therapyalso comes up against the instability of the molecule, which is highlydependent upon the state of glycosylation of the molecule (Saraneva etal., 1995), which is very difficult or impossible to obtain with theexpression systems used.

IFN-γ is a glycoprotein of 133 to 143 amino acids depending on thespecies, which is active in the homodimer form. It acts on the targetcells by stimulating a transmembrane receptor, doing so while respectinga species barrier. This species specificity is based on the specificrecognition between the outer (or extracellular) portion of the IFN-γreceptor and the N-terminal portion of the cytokine.

The association of IFN-γ and its receptor leads to a dimerization ofthis receptor and regulates the cytoplasmic association of tyrosineJanus kinase 2 (JAK 2) with the alpha chain of the receptor. Thisresults in a trans- and/or an autophosphorylation of JAK 1 and JAK 2.This represents the first steps in the activation cascade of thetransduction pathway of the signal associated with the stimulation bythis cytokine, resulting in biological activities such as the inductionof the expression of class II CMH molecules, Fc-γ receptors, andadhesion molecules such as VCAM-1.

However, a biological activity independent of the species barrier isobserved when the cytokine is delivered inside target cells (inliposomes (Fidler et al., 1985)), or introduced by micro-injection(Smith et al., 1990), or transfection (Sanceau et al., 1987). Theseresults appeared to suggest the existence of an intracellular activationpathway.

This second activation pathway quite probably takes place afterinteraction of the C-terminal portion of IFN-γ (sequence 95-133 in thecase of murine IFN-γ), with the alpha chain of the IFN-γ receptor. Thissequence has a binding site which is independent of the species, locatedat the level of residues 253-287 of the alpha chain of the murine IFN-γreceptor which is of strong affinity (Szente and Johnson, 1994). Thisintracytoplasmic binding site is located close to the cell membrane andto the JAK 2 tyrosine kinase binding site. This second recognition stepappears to be essential to the biological function of this cytokine: theinteraction of the C-terminal peptides of IFN-γ with the cytoplasmicportion of the IFN-γ receptor increases the binding of JAK 2 with thealpha chain of the receptor (Szente et al., 1995), which results in anactivation of the signal transduction pathway.

These observations supported and explained previous observationsregarding the capacity to activate the IFN-γ receptor of murinemacrophages with vectorized human IFN-γ. The probable physiologicalmechanism of action of IFN-γ is thus thought to involve aninternalization of the complex formed between IFN-γ and its receptor,following the first recognition step.

Another mechanism may be envisaged, which is thought to correspond to anintracrine stimulation, in the course of which the IFN-γ-producing cellswould be autostimulated by the IFN-γ produced inside the cell, withoutan autocrine activity being necessary via the extracellular portion ofthe cytokine receptor.

In vitro, the treatment of phagocytic cells of the murine P388D1 cellline (macrophage monocytes) with murine (sequence 95-133) or human(sequence 95-134) IFN-γ peptides for 24 hours at high concentration (100μM) can induce expression of the class II CMH receptor. The highconcentration required can be explained by the low degree of penetrationof the peptide across the cell membrane (by means of pinocytosisactivity of the cell studied), or by an inadequate conformation of thepeptide, or by a combination of the two phenomena (Szente.et al., 1994).

More recently, Szente identified the structural elements involved in theagonist activity of the C-terminal peptide, and observed that an α helixcomprising the unit RKRKR is essential for binding to the cytoplasmicdomain of the IFN-γ receptor and for inducing the biological activity(Szente et al., 1996).

Only phagocytic cells have a relative sensitivity to the C-terminalpeptide of IFN-γ as used by Szente. These observations are offundamental interest since they have made it possible to support thehypothesis of the intracrine activity of this cytokine, but they do notpropose a product which can be used as such: the biological activityobserved with the unmodified peptide moreover did not have the completespectrum of activity of IFN-γ. In particular, Szente did not obtaininduction of the antiviral activity considered as the signature of theIFN-γ activity, and which is used to assay the activity of productionbatches of this cytokine, since the cells used to carry out the standardtest have no phagocytic activity.

The therapeutic use of peptides corresponding to the C-terminal portionof mammalian IFN-γ, such as the above-mentioned murine peptide 95-133 orhuman peptide 95-134, would be particularly advantageous since it wouldallow an induction of the biological activity observed in the case ofusing the whole IFN-γ, by direct binding of the said peptide to theIFN-γ intracellular receptor, without passing via the intermediate stepof recognition of the extracellular receptor of this IFN-γ, while at thesame time respecting a physiological mechanism of activation ifreference is made to an intracrine activity, and thus capable ofexhibiting a complete agonist nature.

Specifically, as has been seen above, whole IFN-γ binds first to anextracellular receptor which is specific to a given species. Next, theIFN-γ appears to be internalized inside the cell, and might react withthe intracellular portion of the said receptor, which, as has beenmentioned above, does not appear to be specific to a given species.

Consequently, the use of the above-mentioned peptides corresponding tothe C-terminal portion of mammalian IFN-γ would make it possible tosolve this species-barrier problem, and thus to use a peptidecorresponding to the C-terminal portion of the IFN-γ of a given mammal,in the context of treating all mammals. Furthermore, the direct cellularinternalization of these peptides, thus avoiding the step of recognizingthe extracellular receptor, would have the advantage of limiting theresidence time of the peptide outside the cells, and thus ofcontributing towards limiting the risks of degradation of this peptide.

However, as has been seen above, the low degree of penetration of theabove-mentioned murine IFN-γ peptide 95-133 observed in vitro inmacrophages does not make it possible to envisage a therapeutic use ofthe above-mentioned peptides, since the main target cells of thesepeptides, in particular including the antigen-presenting cells, or thecytotoxic or helper T cells, have at best only a pinocytosis activity,of which it may be deduced, from the above-mentioned 1994 article bySzente et al., that it is insufficient for rapid and efficientinternalization of the said peptide.

The possibility of vectorizing peptides inside living cells by modifyingthem with a lipid chain has already been the subject of studies. Thecomparison of a series of lipopeptides derived from a pseudosubstratesequence of protein kinase C allowed the Inventors to demonstrate thatthe modification of peptides with an N- or C-terminal palmitoyl-lysinegroup leads to the production of a vector which is efficient in terms ofcytoplasmic addressing (Loing et al., 1996). Modification with apalmitoyl-lysine allows a rapid passage of peptides of 9 to 17 residuesinside living cells, which can be observed indirectly by measuring thebiological activity (Thiam et al., 1997) or indirectly by means ofexperiments performed by optical, confocal or electron microscopy.

The present invention derives from the demonstration by the inventors ofthe fact that lipopeptides containing the above-mentioned peptidescorresponding to the C-terminal portion of mammalian IFN-γ allows thesaid peptides to penetrate into cells, independently of a phagocyticactivity, while at the same time conserving their biological activity.The experiments carried out by the Inventors have proved that it wasimpossible to observe this biological activity on non-phagocytic cells,when the above-mentioned peptide 95-133 is used at the same doses as thelipopeptide comprising this peptide 95-133. It was impossible topredict, firstly, that combining a simple lipid chain with such a largepeptide (which is markedly larger in size than those described above inthe article by Thiam et al., 1997) would allow this peptide to cross thecell membrane, and, secondly, that this lipid chain would not prevent,in particular by means of a masking phenomenon, the binding of the saidpeptides to the intracellular portion of mammalian IFN-γ receptors.

The main object of the present invention is thus to provide novellipopeptide compounds allowing an efficient penetration of peptidescorresponding to the C-terminal portion of mammalian IFN-γ into thetarget cells of IFN-γ.

In this respect, the present invention makes it possible to providecompounds that are agonists of the total or partial activity of IFN-γ(i.e. compounds which mimic at least one of the biological orpharmacological activities of IFN-γ), and whose activity wasdemonstrated in vivo for the first time by the Inventors on animals, thesaid compounds being able to be used in human or animal therapy.

A further object of the present invention is to provide novel laboratoryreagents, as well as novel pharmaceutical compositions, comprising theabove-mentioned lipopeptides.

A further object of the invention is to provide novel pharmaceuticalcompositions having the advantage over recombinant IFN-γ of exhibitingfewer side effects than the latter, in particular since the number oftimes the said pharmaceutical compositions are taken is markedly smallerthan that for IFN-γ, and since the delay of action of these compositionsis much shorter than that for IFN-γ.

Furthermore, the pharmaceutical compositions of the invention have theadvantage of being able to be stored for markedly longer periods thanrecombinant IFN-γ, and of allowing this under markedly less restrictiveconditions since there is nothing to fear from a sporadic breakdown oftheir refrigeration conditions and they can thus, in this respect, bestored at ambient temperature.

The invention relates to any lipopeptide which is characterized in thatit comprises:

a peptide portion capable of binding to the intracellular portion of theIFN-γ receptors, but which cannot bind to the extracellular portion ofthe said receptors, the said peptide portion comprising:

the peptide sequence consisting of from about 30 to about 50 of the lastcontiguous amino acids of the C-terminal end of mammalian interferon-γ(IFN-γ), of which, where appropriate, the last 3 to 20 amino acids havebeen suppressed,

or any fragment, in particular of from about 5 to about 30 amino acids,of the above-mentioned peptide sequence of the C-terminal end ofmammalian IFN-γ,

or any peptide sequence derived from the above-mentioned peptidesequence of the C-terminal end of IFN-γ, or of an above-mentionedfragment,

and one or more lipophilic portions, advantageously chosen from thosecomprising:

a linear or branched, saturated or unsaturated C4 to C20hydrocarbon-based chain,

or a steroid group, where appropriate linked to the above-mentionedhydrocarbon-based chain,

the said lipophilic portions optionally being combined with a shortvector peptide (in order thus to form vector lipopeptide units)comprising one or more functions that are ionized at physiological pH,and a function for covalently bonding the said hydrocarbon-based chainand/or the said steroid group.

In the text hereinabove and hereinbelow, the expression “lipophilicportion” means any water-insoluble lipophilic molecule which, when boundto the peptide portion defined above, allows a passive intracellularpassage of the lipopeptide obtained, by virtue of the hydrophobicproperties of the said molecule. The lipopeptide resulting from thebinding of the lipophilic portion to the peptide portion isadvantageously water-soluble.

The hydrocarbon-based chain of the lipophilic portions is preferablychosen from those of:

palmitic acid,

oleic acid,

linoleic acid,

linolenic acid.

Preferably also, the steroid group of the lipophilic portion(s) ischosen from cholesterol derivatives such as cholest-5-enyl-3-oxyaceticacid or cholest-5-enyl-3-oxycarbonic acid.

The invention relates, more particularly, to any lipopeptide asdescribed above which is characterized in that the lipophilic portion(s)is (are) covalently bonded to one or more amino acids of the peptideportion.

It is advantageous if the Lipohile portion(s) (is) are bonded covalentlyto the αNH₂ or εNH₂ function of a lysine located at the N-terminal orC-terminal position of the peptide portion, or to the thiol function ofa cysteine, or to any amino, alcohol or thiol function optionally addedto the peptide with a simple spacer.

In this respect, the invention relates, more particularly, to anylipopeptide as defined above in which the lipophilic portion(s) is (are)represented by an N^(α)-acetyl-Lysine N^(ε)(palmitoyl) group (alsodenoted by the abbreviation Ac-K(Pam)).

The present invention also relates to lipopeptides resulting from acovalent association between a vector lipopeptide unit, as definedabove, which ensures vectorization across a cell membrane, and afunctional unit derived from one of the above-mentioned mammalian IFN-γsequences. The vector lipopeptide unit preferably corresponds to a shortsequence comprising functions that are ionized at physiological pH (Arg,Lys, Asp or Glu), and a lipid (or lipophilic) portion as describedabove. The covalent association between the vector lipopeptide unit andthe functional unit can be an amide bond, or a non-peptide bondresulting from a simple chemical ligation, obtained by virtue of thereactivity of the thiol (thio ether, thio ester or disulphide) functionor of an aldehyde function with a weak base (thiazolidine, oxime,hydrazone). Examples of such ligations, which are conventional to thoseskilled in the art, are described in the review by Tam and Spetzler,1995.

By the definition given above of the peptide portion of the lipopeptidesof the invention, that is to say “the peptide sequence consisting offrom about 30 to about 50 of the last contiguous amino acids of theC-terminal end of mammalian interferon-γ (IFN-γ), of which, whereappropriate, the last 3 to 20 amino acids have been suppressed”, thismeans any peptide sequence consisting of from about 30 to about 50 ofthe last contiguous amino acids of the C-terminal end of mammalianIFN-γ, as well as any peptide sequence consisting of from about 30 toabout 50 of the last contiguous amino acids of the C-terminal end ofmammalian IFN-γ fragments, these fragments corresponding to the varioustypes of mammalian IFN-γ of which the last 3 to 20 amino acids have beensuppressed.

The above-mentioned peptide sequence consisting of from about 30 toabout 50 of the last contiguous amino acids of the C-terminal end ofmammalian IFN-γ (also denoted hereinbelow as the sequence correspondingto the IFN-γ C-terminal portion), as well as the above-mentionedfragments and sequences derived from this sequence, advantageouslycontain a sequence of the type XKRYR in which X represents R, G, I, F orK, and Y represents K or R.

Advantageously also, the said peptide sequence corresponding to theIFN-γ C-terminal portion, as well as the above-mentioned fragments andsequences derived from this sequence, specifically recognize theintracellular portion of mammalian IFN-γ receptors and, in this respect,have at least one of the biological and pharmacological properties ofmammalian IFN-γ, and are referred to as being agonists of the total orpartial activity of mammalian IFN-γ.

By way of illustration, the peptide sequences corresponding to the IFN-γC-terminal portion, in the above-mentioned lipopeptides, or thefragments of these sequences, or the sequences derived from thesesequences or fragments, are further characterized in that theyspecifically recognize:

the intracellular portion of the human IFN-γ receptor delimited by theamino acids located in positions 252 and 291 of the peptide sequence ofthe said receptor,

or any intracellular portion of the IFN-γ receptors of mammals otherthan man, in particular the intracellular portion of the murine IFN-γreceptor delimited by the amino acids located in positions 253 and 287of the peptide sequence of the said receptor.

As has been mentioned above, the peptide portion of the above-mentionedlipopeptides of the invention does not recognize the extracellularportion of the mammalian IFN-γ receptor with IFN-γ.

In this respect, the said peptide portion of the lipopeptides of theinvention is such that, when it contains the N-terminal end of mammalianIFN-γ, at least one of the first 20 amino acids of this N-terminal endis suppressed or substituted with a natural or non-natural amino acid,such that the said N-terminal end thus modified is incapable ofrecognizing and binding to the extracellular IFN-γ receptor.

The peptide portion of the above-mentioned lipopeptides of the inventionadvantageously does not comprise the N-terminal end of mammalian IFN-γ,which is required for the specific recognition of the extracellularportion of the mammalian IFN-γ receptor with IFN-γ.

The peptide portion of the above-mentioned lipopeptides of the inventionis preferably such that it does not comprise the sequence delimited, onthe one hand, by the amino acid located in position 1, and, on the otherhand, by the amino acid located in position 14 of the peptide sequencesof mammalian IFN-γ (in particular of the IFN-γ peptide sequencesindicated below).

The invention relates, more particularly, to any lipopeptide as definedabove which is characterized in that the peptide portion comprises atleast one of the following peptide sequences:

the sequence delimited by the amino acids located in positions 95 and134:

of the following human IFN-γ peptide sequence:

SEQ ID NO: 2

of the following bovine IFN-γ peptide sequence:

SEQ ID NO: 3

of the following IFN-γ peptide sequence from the monkey Callithrixjacchus:

SEQ ID NO: 4

of the following IFN-γ peptide sequence from the monkey Cercocebustorquatius atys:

SEQ ID NO: 5

of the following dog IFN-γ peptide sequence:

SEQ ID NO: 6

of the following cat IFN-γ peptide sequence:

SEQ ID NO: 7

of the following deer IFN-γ peptide sequence:

SEQ ID NO: 8

of the following chicken IFN-γ peptide sequence:

SEQ ID NO: 9

of the following horse IFN-γ peptide sequence:

SEQ ID NO: 10

of the following macaque IFN-γ peptide sequence:

SEQ ID NO: 11

of the following pig IFN-γ peptide sequence:

SEQ ID NO: 12

of the following rabbit IFN-γ peptide sequence:

SEQ ID NO: 13

of the following sheep IFN-γ peptide sequence:

SEQ ID NO: 14

of the following marmot IFN-γ peptide sequence:

SEQ ID NO: 15

of the following IFN-γ peptide sequence of Meriones unguiculatus:

SEQ ID NO: 16

the sequence delimited by the amino acids located in positions 95 and133 or 132:

of the following murine IFN-γ peptide sequence:

SEQ ID NO: 17

of the following rat IFN-γ peptide sequence:

SEQ ID NO: 18

the sequence delimited on the N-terminal side by an amino acid locatedat one of positions 113 to 121, and on the C-terminal side by the aminoacid located in position 132 of the murine IFN-γ peptide sequencerepresented above.

The invention also relates to any lipopeptide as defined above which ischaracterized in that the COOH function of the C-terminal amino acid issubstituted with a group which is resistant to the organism'sexopeptidases, in particular with a carboxamide group.

The invention relates, more particularly, to any lipopeptide as definedabove, the peptide sequence of which is that:

delimited by the amino acids located in positions 95 and 134 of thehuman IFN-γ peptide sequence represented above,

or delimited by the amino acids located in positions 95 and 133 or 132of the murine IFN-γ peptide sequence represented above,

or delimited on the N-terminal side by an amino acid located in one ofpositions 113 to 121, and on the C-terminal side by the amino acidlocated in position 132, of the murine IFN-γ peptide sequencerepresented above, and more particularly that delimited by the aminoacids located in positions 113 and 132 of the said IFN-γ peptidesequence,

the COOH function of the C-terminal amino acid of the above-mentionedpeptide sequences being, where appropriate, substituted with a groupwhich is resistant to the organism's exopeptidases, in particular with acarboxamide group.

Lipopeptides that are preferred in the context of the present inventionare the following:

the lipopeptide whose sequence is delimited by the amino acids locatedin positions 95 and 134 of the human IFN-γ peptide sequence representedabove, corresponding to the following formula:

SEQ ID NO 19

the lipopeptide whose sequence is delimited by the amino acids locatedin positions 95 and 132 of the murine IFN-γ peptide sequence representedabove, corresponding to the following formula:

SEQ ID NO 20

the lipopeptide whose sequence is delimited by the amino acids locatedin positions 113 and 132 of the murine IFN-γ peptide sequencerepresented above, corresponding to the following formula:

SEQ ID NO 21.

The expression “peptide sequence derived from the above-mentionedpeptide sequence of the C-terminal end of IFN-γ, or of a fragment of thelatter”, in the context of the lipopeptides of the invention, means anysequence derived:

by substitution and/or suppression and/or addition of one or more aminoacids, of the above-mentioned sequence or fragment, and/or

by modification of at least one peptide linkage —CO—NH— of the peptidechain, of the above-mentioned sequence or fragment, in particular byintroducing a linkage of the retro or retro-inverso type, and/or

by substitution of at least one amino acid of the peptide chain of theabove-mentioned sequence or fragment, with a non-protein-generatingamino acid,

the said derived sequence specifically recognizing the intracellularportion of the mammalian IFN-γ receptors and, in this respect, having atleast one of the biological or pharmacological properties of mammalianIFN-γ, in particular at least one of the properties listed above.

The expression “sequence derived by introducing a retro-inverso linkage”should be understood as meaning any peptide analogue of theabove-mentioned sequence or fragment, the said analogue consisting of apeptide chain in which at least one of the residues is, on the one hand,linked to at least one neighbouring residue via an —NH—CO— linkage, andis, on the other hand, of opposite chirality to that of this sameaminoacyl residue in the peptide chain of the parent peptide (that is tosay of the above-mentioned sequence or fragment).

The expression “sequence derived by introducing a retro linkage” shouldbe understood as meaning any peptide analogue of the above-mentionedsequence or fragment, the said analogue consisting of a peptide chain inwhich at least one of the residues is linked to at least oneneighbouring residue via an —NH—CO— linkage, the chirality of all of theaminoacyl residues involved in at least one —NH—CO— linkage beingconserved relative to the corresponding residues of the peptide chain ofthe parent peptide.

It goes without saying that the —CO—NH— and —NH—CO— linkages should beconsidered, in the text hereinabove, in the direction of the parentpeptide chain going from the amino-terminal (N-terminal) end to thecarboxy-terminal (C-terminal) end.

In the text hereinabove, the expression “protein-generating amino acid”means any amino acid forming part of the constitution of a naturalprotein or peptide.

In contrast to the preceding definition, the expression“non-protein-generating amino acid” means any amino acid which does notform part of the constitution of a natural protein or peptide. Theexpression “non-protein-generating amino acid” more particularly meansany amino acid whose carbon bearing the side chain R, i.e. the group—CHR—, located between —CO— and —NH— in the natural peptide chain, isreplaced with a unit which does not form part of the constitution of anatural protein or peptide.

The invention relates, more particularly, to the derived sequences asdescribed above, characterized in that at least one of the —CO—NH—peptide linkages of the peptide chain of the parent peptide is replacedwith a linkage other than the —CO—NH— linkage, the said other linkagebeing chosen in particular from the following:

—CH₂—NH— (methyleneamino);

—CH₂—CH₂— (carba);

—CO—CH₂— (ketomethylene);

—CH₂—O— (methylenoxy);

—CHOH—CH₂— (hydroxyethylene);

—CHOH—CHOH— (dihydroxyethylene);

—CH═CH— (E or Z olefin);

—CHCN—NH— (cyanomethyleneamino);

—S—CH₂— (thiomethylene);

—CH₂—S— (methylenethio);

—CS—NH— (thio amide);

—PO₂—NH— (phosphonamide);

—CHOH— (hydroxymethylene);

—NH—CO—NH— (urea);

—CH₂—CO—NH— (homologation);

—CHOH—CH₂—NH— (hydroxyethylene amino);

—CO—NH—NH— (hydrazino).

The peptide sequences corresponding to the IFN-γ C-terminal portion, inthe above-mentioned lipopeptides, or the fragments of these sequences,or the sequences derived from these sequences or fragments, as definedabove, are advantageously IFN-γ agonists and have at least onebiological activity of the mammalian IFN-γ type, that is to say at leastone of the following properties:

1) as regards the biological properties:

an immunoadjuvant effect,

an antiviral effect,

an immunomodulatory effect, in particular by:

stimulating the cellular production of cytokines,

action on the presentation of antigens, in particular:

by increasing the expression of class I HLA genes, and of theβ2-microglobulin gene; this results in an increase in the possibilitiesof presentation of antigens foreign to the CD8+lymphocytes,

by increasing or inducing the expression of class II HLA genes, and ofthe invariant chain; this results in an increase in the possibilities ofpresentation of antigens foreign to the CD4+lymphocytes,

action on the cellular adhesion phenomena, by stimulating the synthesisand expression of the VCAM-1 protein at the surface of the various cells(macrophages, endothelial cells), thus promoting the immunologicalphenomena of cellular cooperation,

increasing the expression of the complement proteins,

a maturation effect on the B lymphocytes,

a maturation effect on the cytotoxic T lymphocytes,

an activation effect on the NK cells (increase of their cytotoxicactivity) pand an inductive effect on the generation of LAK cells(lymphokine-activated killer cells),

an effect of increasing the cytotoxic possibilities of the monocytic andmegakaryocytic lines, in particular by:

increasing the synthesis of TNFα,

stimulating the production of oxygenated free radicals,

producing nitric oxide (NO—),

inducing the production of tryptophan-derived mediators, such aspicolinic acid,

increasing the release of lysosomal enzymes,

an action on the inflammatory reaction,

an effect on fibrinogenesis (anti-fibrinogenic) and on the fibrinolysisand haemostasis system,

anti-infectious effects;

2) as regards the pharmacological properties:

an effect of reducing the incidence of infectious complications inpatients suffering from chronic granulomatosis,

effects in infectious immunotherapy, in particular:

an antibacterial effect,

an antifungal effect,

an antiparasitic effect, in particular in the case of leishmaniasis,toxoplasmosis and malaria,

an anithelmintic effect,

effects against viral infections,

effects against atopy and allergic pathologies, in particular pulmonaryhypereosinophilic allergies, or skin allergies (dermatitis),

effects against autoimmune diseases,

anti-cancer effects, in particular in the case of cancer of the kidneys,cutaneous T lymphomas, chronic myeloid leukaemia, cancer of the ovariesand mesotheliomias.

The present invention also relates to micelles or micro-aggregates ofone or more different lipopeptides defined above.

The said micelles or micro-aggregates are advantageously less than about1 μm in size.

The micelles or micro-aggregates according to the invention arepreferably as obtained by dispersing the said lipopeptides in anapproximately 80% concentrated acetic acid solution, or any othersolvent capable of ensuring molecular dispersion of the lipopeptides insolution.

The invention also relates to any pharmaceutical composition which ischaracterized in that it comprises one or more lipopeptides, whereappropriate in the form of micelles, as described above, and incombination with a vehicle in the context of a physiologicallyacceptable pharmaceutical formulation.

The invention relates, more particularly, to the use of lipopeptides,where appropriate in the form of micelles, as described above, for thepreparation of a medicinal product intended for treating and, whereappropriate, preventing pathologies against which IFN-γ is liable toact, in particular against the pathologies listed above in the contextof the pharmacological properties of IFN-γ.

The lipopeptides, where appropriate in the form of micelles, mentionedabove are advantageously used for the preparation of an antiviralmedicinal product intended for treating viral pathologies such as AIDS,conditions caused by papillomaviruses (in particular certain uterinecancers), the various forms of hepatitis, including hepatitis B, or thevarious non-A non-B hepatitides.

Advantageously also, the above-mentioned lipopeptides, where appropriatein the form of micelles, are used for the preparation of a medicinalproduct for treating bacterial or viral pulmonary infections, such astuberculosis or pneumocystosis, or for treating bacterial or viralinfections of the ORL area (and more particularly of thebucccopharyngeal area).

The above-mentioned pharmaceutical compositions are preferably in apharmaceutical form which allows a high concentration of activeprinciple to be obtained in an area of micro-diffusion around the siteof parenteral, intramuscular, subcutaneous or intradermal injection, orfrom a contact surface (intra-pulmonary aerosol or nebulizate,sublingual, transmucous or percutaneous route), or alternatively in aform which can be applied topically, in particular in the form of acream or ointment.

The preferred dosages of the above-mentioned pharmaceutical compositionsfor a treatment such as a treatment with IFN-γ should be related to thedosages used in the case of recombinant IFN-γ, given that 2 to 3 μg oflipopeptide according to the invention correspond to 1 IU of IFN-γ. Forexample, the prescribed doses of recombinant IFN-γ in the context of aclinical trial carried out on patients suffering from metastatic renalcarcinoma are 1 mg/m² i.v. per day for 5 days, one week in two, for onemonth (approximately 4 to 5×10⁶ IU per dose, depending on the specificactivity of the batch, set at around 0.2 ng/1 IU (Mani S., Poo W. J.,Am. J. Clin. Oncol., 1996, 19: 2, pp. 149-153)).

As immunoadjuvant combined with a vaccine, as described below, the doseof lipopeptide according to the invention is advantageously from about125 to about 500 μg/ml (on average about 250 μg/ml), for injectedvolumes of about from 0.5 to 3 ml in man, or in veterinary application.

As an IFN-γ analogue, used alone for its immunostimulatory, anti-canceror anti-infectious properties, the preferred doses of the lipopeptide ofthe invention, when it is administered by inhalation or via theintranasal route, of solutions at concentrations of about 25 to 50 μM,are thus from about 250 to 500 μg/ml, i.e. about 1.5 to 3 mg perdose/per day in man (as a general rule, at a rate of 3 times a week, for6 months).

The invention also relates to any composition characterized in that itcomprises one or more lipopeptides, where appropriate in the form ofmicelles, as described above, in combination with:

one or more peptides or lipopeptides containing one or more epitopesspecifically recognized by the cytotoxic T lymphocytes (also referred toas CTLs), and capable of activating the latter (also referred to as CTLepitopes or CD8+epitopes), and/or

one or more peptides or lipopeptides containing one or more epitopesspecifically recognized by the helper T lymphocytes (also referred to asHTLs), and capable of activating the latter (also referred to as HTLepitopes or CD4⁺ epitopes), and/or

one or more peptides or lipopeptides containing one or more B epitopesspecifically recognized by antibodies directed against the latter.

The invention relates, more particularly, to any composition as definedabove which is characterized in that the said CD8⁺ epitopes are:

those characteristic of tumour cells, such as:

the epitopes of chronic myeloid leukaemia (in particular those listed inTable 1),

the epitopes of protein p53 (in particular those listed in Table 2),

melanoma epitopes (in particular the human melanoma epitopes listed inTable 3), and more particularly the epitopes of the human melanomamelan-A/mart-1 antigen

the epitopes of tumours resulting from mutations (in particular thoselisted in Table 4),

antigens common to various tumours, such as those listed in Table 5),

those characteristic of viral proteins, such as:

the epitopes of hepatitis B virus (HBV) proteins,

the epitopes of AIDS virus (HIV) proteins (in particular those listed inTable 6),

the epitopes of human papillomavirus (HPV) proteins, in particular theHPV E₆ or E₇ proteins (in particular those listed in Table 7).

The invention relates, more particularly, to any composition asdescribed above which is characterized in that it contains, as CD4⁺epitopes, multiple epitopes such as the tetanus toxin peptideTT(830-846) (Panina-Bordignon et al., 1989), the influenzahaemagglutinin HA(307-319) (Krieger et al., 1991), PADRE (Alexander etal., 1994), the HIV-1 45-69 NEF peptide (Estaquier et al., 1992), theLSA3 peptide of Plasmodium falciparum, which is the agent responsiblefor malaria (Ben Mohamed et al., 1997).

The invention relates, more particularly, to any composition asdescribed above which is characterized in that it contains, as Bepitope, one of the epitopes of a protein associated with an allergicreaction, such as the house dust allergens, in particular of thepeptides of Dermatophagoides pteronyssinus (peptides 52-71, 117-133,176-187 or 188-199) or of Dermatophagoides farinae.

The invention also relates to any lipopeptide as defined above whichcomprises one or more above-mentioned CD8⁺ and/or CD4⁺ and/or Bepitopes, the said epitopes being covalently bonded to the lipophilicportion of the said lipopeptide, and/or to the above-mentioned peptidesequence of the C-terminal end of mammalian IFN-γ, or to fragments orsequences derived therefrom.

The lipopeptides described above are preferably such that their peptideportion comprises one or more above-mentioned CD8⁺ and/or CD4⁺ and/or Bepitopes, the said epitopes being covalently bonded (directly or via asequence of about 2 to 5 amino acids) to the above-mentioned peptidesequence of the C-terminal end of mammalian IFN-γ, or to fragments orsequences derived therefrom. The linkages between the said epitopes andthe said peptide sequence of the C-terminal end are preferably peptidelinkages, or any of the linkages resulting from simple ligations asmentioned above.

The invention also relates to micelles or micro-aggregates of one ormore different lipopeptides which comprise one or more covalently bondedCD8⁺ and/or CD4⁺ and/or B epitopes, as defined above.

As above, the said micelles or micro-aggregates are advantageously lessthan about 1 μm in size, and are preferably obtained by dispersing thesaid lipopeptides in an approximately 80% concentrated acetic acidsolution.

The invention also relates to any pharmaceutical composition or vaccinewhich is characterized in that they comprise:

a composition of one or more lipopeptides, where appropriate in the formof micelles, in combination with one or more CD8⁺ and/or CD4⁺ and/or Bepitopes, the said composition being as defined above, and/or

one or more lipopeptides, where appropriate in the form of micelles,comprising one or more covalently bonded CD8⁺ and/or CD4⁺ and/or Bepitopes, as defined above, and optionally one or more lipopeptides asdefined above, containing only the above-mentioned peptide sequence ofthe C-terminal end of IFN-γ, where appropriate in the form of micelles,

in combination with a vehicle in the context of a physiologicallyacceptable pharmaceutical formulation.

The above-mentioned pharmaceutical compositions comprising epitopes arein a pharmaceutical form which allows a high concentration of activeprinciple to be obtained in a microdiffusion area around the site ofparental, intramuscular, subcutaneous or intradermal injection, or froma contact surface (intrapulmonary aerosol or nebulizate, sublingual,transmucous or percutaneous route), or alternatively in a form which canbe applied topically, in particular in the form of a cream or ointment.

The preferred dosages are as defined above.

A subject of the invention is, more particularly, the use:

of a composition of one or more lipopeptides, where appropriate in theform of micelles, in combination with one or more CD8⁺ and/or CD4⁺and/or B epitopes, the said composition being as defined above, or

of one or more lipopeptides, where appropriate in the form of micelles,comprising one or more covalently bonded CD8⁺ and/or CD4⁺ and/or Bepitopes, as defined above,

for the preparation of a medicinal product or vaccine for inducing aspecific immune response against the antigens corresponding to the saidepitopes, more particularly in the context of treating and, whereappropriate, preventing pathologies which can be controlled byactivating CTL and/or HTL by means, respectively, of the said CD8⁺epitopes linked to the class I CMH molecules, and/or of the CD4⁺epitopes linked to the class II CMH molecules, at the surface ofantigen-presenting cells, and/or for the preparation of a medicinalproduct or vaccine for re-orienting the immune response by antibodiesdirected against B epitopes, and more particularly directed against anallergen.

The invention relates, more particularly, to the above-mentioned use:

of a composition as described above of one or more lipopeptides, whereappropriate in the form of micelles, in combination with one or moreCD8⁺ epitopes,

or of one or more lipopeptides as defined above, where appropriate inthe form of micelles, comprising one or more covalently bonded CD8⁺epitopes,

in which the epitopes are those characteristic:

of tumour cells, as described above, for the preparation of ananti-tumour medicinal product, intended for the treatment of tumourpathologies such as chronic myeloid leukaemia, or melanoma, or

of viral proteins, as described above, for the preparation of amedicinal product or vaccine intended for preventing and, whereappropriate, treating viral pathologies such as AIDS, conditions causedby papillomaviruses (in particular certain uterine cancers), the variousforms of hepatitis, including hepatitis B, or the various non-A non-Bhepatitides.

The invention relates, more particularly, to the above-mentioned use:

of a composition as described above of one or more lipopeptides, whereappropriate in the form of micelles, in combination with one or moreCD4⁺ epitopes,

or of one or more lipopeptides as defined above, where appropriate inthe form of micelles, comprising one or more covalently bonded CD4⁺epitopes,

in which the said CD4⁺ epitopes are multi-specific epitopes capable ofpotentiating the immune response against any other antigen in anunselected population, and are in particular those characteristic oftetanus toxin TT(830-846), the influenza haemagglutinin HA(307-319),PADRE, the HIV-1 45-69 NEF peptide, and the LSA3 peptide of Plasmodiumfalciparum, mentioned above,

for the preparation of a medicinal product or vaccine for potentiatingthe immune response against any other antigen, in particular in thecontext of viral or parasitic pathologies.

The invention relates, more particularly, to the above-mentioned use:

of a composition as described above of one or more lipopeptides, whereappropriate in the form of micelles, in combination with one or more Bepitopes,

or of one or more lipopeptides as defined above, where appropriate inthe form of micelles, comprising one or more covalently bonded Bepitopes,

in which the epitopes are those characteristic of proteins associatedwith an allergic reaction, such as the B epitopes corresponding to theallergens of house dust, in particular the peptides of Dermatophagoidespteronyssinus (peptides 52-71, 117-133, 176-187 or 188-199), or ofDermatophagoides farinae, for the preparation of a medicinal product orvaccine intended for preventing and, where appropriate, treatingallergic pathologies such as allergic asthma.

The invention also relates to the use of any lipopeptide or of anylipopeptide composition, as described above, in the context of carryingout methods for the in vitro (or ex vivo) treatment of cells of thehuman or animal body, the said method comprising a step of taking thesaid cells from the human or animal, who or which is healthy or requiresa treatment, followed by a step of treating the said cells by incubatingthem with a lipopeptide or a lipopeptide composition according to theinvention, and a step of administering the cells thus treated to thepatient from whom they were taken, or to any other patient requiringsuch a treatment.

The invention also relates to the use of lipopeptides as defined above,where appropriate in the form of micelles, as laboratory reagents, inparticular:

to reproduce the cellular activation effects of IFN-γ, by adding thelipopeptides according to the invention to cells in order to activatethem before carrying out other explorations on these cells,

as immunoadjuvant to test the immune response of a vaccinating principleunder study,

as immunomodulator, that is to say for its ability to polarize theimmune response.

The invention also relates to any peptide whose sequence is delimited onthe N-terminal side by an amino acid located in one of the positions 113to 121, and on the C-terminal side by the amino acid located in position132, of the murine IFN-γ peptide sequence represented above.

In this respect, the invention relates, more particularly, to thepeptide delimited by the amino acids located in positions 113 and 132 ofthe murine IFN-γ peptide sequence, and corresponding to the sequencebelow:

SEQ ID NO: 1

The invention also relates to any peptide as described above which ischaracterized in that the COOH function of the C-terminal amino acid issubstituted with a group which is resistant to the organism'sexopeptidases, in particular with a carboxamide group.

The above-mentioned peptide sequences used in the context of the presentinvention are advantageously synthesized chemically, in particularaccording to the conventional techniques of solid phase peptidesynthesis described in the experimental section which follows.

As a variant, the above-mentioned peptide sequences can be obtained viagenetic engineering, in particular by transforming suitable host cellswith vectors containing the DNA sequences encoding the said peptidesequences.

The invention will be further illustrated with the aid of the detaileddescription which follows, of the preparation of lipopeptides accordingto the invention, as well as of the studies of their biologicalproperties.

A) Study of the MuL Peptide I—Peptide Synthesis of the MuL LipopeptideAccording to the Invention

The following peptide, also referred to as the Mu peptide, andcorresponding to the sequence delimited by the amino acids located inpositions 95 and 132 of the murine IFN-γpeptide sequence, wassynthesized.

SEQ ID NO 22

The following modifications were carried out on the Mu peptide:

a carboxamide end was introduced at the C-terminus to reinforce thestability with respect to exopeptidases,

the N-terminal end of the peptide was modified with anN^(α)-acetyl-Lysine N^(ε)(palmitoyl) group, to allow penetration of thepeptide across the membrane independently of the cellular activity.

The Mu peptide thus modified was denoted as MuL, and is represented bythe following formula:

(Ac—K(Pam)=N^(α)-acetyl-Lysine N^(ε)(palmitoyl)  SEQ ID NO 20

A control lipopeptide, also referred to as “scrambled”, corresponding tothe MuL lipopeptide in which the order of the amino acids has beenarranged so as to avoid any sequential relationship with the peptide oforigin, was synthesized. This control lipopeptide is obtained from theMuS peptide of the following formula:

SEQ ID NO 23

and was denoted as peptide MuSL Ac—K(Pam); it corresponds to thefollowing formula:

SEQ ID NO 24

Non-lipid analogues of the above-mentioned peptides were alsosynthesized, with the aim of carrying out comparative studies:

Mu peptide: SEQ ID NO 22

MuS peptide: SEQ ID NO 23

Peptide synthesis: The peptides were synthesized on an MBHA resin (0.63mmol/g, Applied Biosystems, Foster City, USA) using the Boc-benzylstrategy (Merrifield, R. B., 1963; Merrifield, R. B., 1986) and the insitu neutralization protocol, using an AB1 430A peptide synthesizer(Foster City, USA). The protected amino acids are obtained fromPropeptide (Vert-le-Petit, France). The side chains are protected asfollows: Arg(Tos), Thr(Bzl), Asp(OcHex), Glu(OcHex), Gln(Trt), Asn(Trt),Lys(2-ClZ), His(Bom). An acetylation was carried out systematicallyafter each recoupling on the N-terminal function, using 10% aceticanhydride and 5% DIEA in CH₂Cl₂.

In order to obtain the lipopeptide, an N-terminal lysine was introducedvia Boc-L-Lys(Fmoc)-OH (France Biochem, Meudon, France). At the end ofthe synthesis, the Fmoc group was removed with 20% piperidine in DMF.The lipopeptide is obtained after selective acylation of the ε-aminogroup of the N-terminal Lys on the peptidyl-resin (palmiticacid/HBTU/DIEA : 4 eq/4 eq/12 eq in DMF for 30 min, ×2). The lipopeptideis cleaved from the resin (dry resin/HF/p-cresol/thiocresol: 1 g/10ml/0.75 g/0.25 g, 1 h 30 min at 0° C.) and lyophilized. The purificationis carried out by several RP-HPLC chromatographies on a C18 Nucleosilcolumn (12.5 mm×500 mm, solvent A: H₂O containing 0.05% TFA; solvent B:MeCN/H₂O: 4/containing 0.05% TFA). The homogeneity is confirmed byRP-HPLC on C3 Zorbax (4.6×250 mm). Using 0.25 mmol of MBHA resin, 140 mg(cumulative yield of 10%) of lipopeptide with a purity of greater than95% are obtained. The identity was confirmed by determining the aminoacid composition after total acid hydrolysis, and by determining themolecular mass by TOF-PDMS (Bio-lon 20 Plasma Desorption MassSpectrometer): [MH⁺] calc.: 4980.9; obs.: 4982.7.

II—Summary of the Biological Results

All the tests mentioned below have in common the use of complete culturemedia, without removing the foetal calf serum, or introducing anyprotease inhibitor in order to be under the optimum cell cultureconditions without, however, attempting to protect the peptideconstructs from possible degradations due to components intrinsic to theculture medium used.

1—Induction of Class II CMH Molecules:

Principle: to check the induction of class II CMH molecules on celllines (P388D1 and WEHI3: murine myelomonocytes) with the above-mentionedpeptides.

Procedure. The cells are cultured the day before at a rate of 3×10⁵cells per well in 24-well culture plates (NUNC). The following day, thecells are stimulated with the various lipopeptide and peptide constructsat a final concentration of 50 μM in 1 ml of medium. After incubationfor 24 hours at 37° C. in an atmosphere saturated with 5% CO₂, the cellsare recovered and incubated for 1 hour at 4° C. with 3 μg of abiotinylated mouse anti-I-Ad monoclonal antibody (Pharmingen, San Diego,USA). After revelation for 30 minutes with FITC streptavidin used at adilution of 1:100 (Sigma, St. Louis, USA), the expression of class IImolecules is determined by flow cytometry.

Results. Table 8 below represents the percentage of class II CMHdetected by flow cytometry, on the various cell lines stimulated for 24hours with the various peptide constructs.

TABLE 8 name of the peptide P388D1 WEH1 untreated cells  3 3 Mu  5 8 MuS14 5 MuL 98 64  MuSL 13 15 

As observed by Szente (Szente et al., 1994), the induction of the classII CMH molecules is at a maximum after stimulation for 24 hours, whereaswith recombinant IFN-γ, it reaches its maximum after 48 hours; thissuggests that the synthetic constructs would effectively allow a fasteractivation of the signal transduction pathway.

Furthermore, the lipopeptide construct is more active than thenon-vectorized peptide at the concentration studied. This is due to thepresence of the grafted palmitic acid, which gives the lipopeptidebetter cytoplasmic addressing. In the light of these results, the studywas extended on cells taken from an animal (Balb/c mouse) in order to beable to evaluate the potential of MuL in ex vivo studies.

2—Induction of the Expression of Class II CMH Molecules and of Fc-γReceptors (Fc-γ R) on Splenocytes and Peritoneal Cells Taken From anAnimal (Balb/c mouse).

Principle: to evaluate the activity of the vectorized agonist on cellstaken from an animal and treated in vitro (in order to assess the valueof a subsequent in vivo study).

Procedure. The splenocytes and peritoneal cells taken from an animal(Balb/c mouse) are re-cultured at a rate of 2.5×10⁶ splenocytes and 106peritoneal cells per well in 24-well plates (NUNC) stimulated with 50 μMof the different peptides in a final volume of 1 ml. After stimulationfor 24 hours, the cells are labelled with the anti IA^(d) monoclonalantibody described in the preceding paragraph, according to the sameprotocol. To detect the Fc-γ R, the cells are labelled with 1 μg of arat anti Fc-γ R monoclonal antibody (Pharmingen, San Diego, USA). Next,the cells are incubated for ½ hour with a rat anti IgG biotinylatedpolyclonal antibody. The revelation is then carried out with FITCstreptavidin used at a dilution of 1:100 (Sigma, St. Louis, USA). Theexpression of Fc-γ R is determined by flow cytometry.

Results. The percentage of IA^(d) and Fc-γ R detected by flow cytometryon splenocytes and peritoneal cells taken from the animal is representedin Table 9 below.

TABLE 9 splenocytes peritoneal cells name of the peptide RFc (%) II CMH(%) RFc (%) II CMH (%) untreated cells 9 10 6 5 Mu 7 12 8 13 MuS 8 9 8 6MuL 75 83 45 60 MuSL 15 14 20 13

A marked increase of IA^(d) and of Fc-γ R may be observed on the cellstreated with MuL, whereas its scrambled lipopeptide control has nonotable activity. Furthermore, the activity of MuL is markedly higherthan that of Mu; this again confirms the undeniable advantage affordedby adding palmitic acid. These results show that the activity observedis not limited to one or two types of cell lines, but that it is alsoobserved on cells taken from an animal, and this being independent oftheir cellular function.

3—Confirmation of the Involvement of the IFN-γ Receptor

Principle. to confirm that the biological activity observed waseffectively associated with a stimulation of the IFN-γ receptor, werepeated the same experiments, in parallel on splenocytes taken frommice 129 (Wild type: WT) and on cells taken from mice 129 no longerexpressing the alpha chain of the IFN-γ receptor (KO mice).

Procedure. The experimental protocol followed is identical to thatdescribed in paragraph 2 above.

Results. Table 10 below shows the results obtained following stimulationfor 24 hours of cells taken from KO animals for the a chain of the IFN-γreceptor. The expression of IA^(d) and of Fc-γ R is analysed by flowcytometry.

TABLE 10 WT mice KO mice name of the peptide RFc (%) II CMH (%) RFc (%)II CMR (%) untreated cells 1 4 1 3 Mu 1 12 1 3 MuS 1 3 1 3 MuL 23  52 34 MuSL 2 4 3 4

It can be seen that MuL is capable of inducing the expression of IA^(d)and of Fc-γ R on the cells taken from the wild type animal, whereas nosimilar activity is obtained on the cells taken from the animals whichare deficient in respect of the IFN-γ receptor. This provides proof thatthe activity of MuL acts via an interaction with the cytokine receptor,and thereby confirms the specificity of the biological activity observedwith an agonist. Consequently, the vectorized construct (or lipopeptide)induces the expression of II CMH and of Fc-γ R via an interaction withthe receptor for this cytokine.

4—Confirmation of the Intracellular Penetration of the VectorizedAgonist, Onfirmation of the Potential of MuL to Stimulate Human Cells.

Principle: murine IFN-γ is incapable of stimulating human cells, unlessacting intracellularly: the demonstration of a biological activityinduced by the MuL lipopeptide on human cells consequently means thatthe peptide has been able to interact with the internal portion of theIFN-γ receptor, and constitutes indirect proof of the penetration of thelipopeptide into the cytoplasmic compartment.

Procedure. A primary culture of human dermal cells at confluence in a96-well plate (NUNC) is stimulated with the various peptide constructsat a final concentration of 25 and 50 μM (in a final volume of 100 μl)or with human IFN-γ for 24 hours. Next, the expression of VCAM-1adhesion molecules is evaluated by “cell-ELISA”. To do this, the cellsare labelled at 4° C. with 0.5 μg of a mouse anti VCAM-1 monoclonalantibody (Pharmingen, Cambridge, USA). The cells are then fixed withparaformaldehyde and labelled using a peroxidase-coupled goat anti-mouseIg(G, A and M) polyclonal antibody. A revelation with o-phenylenediamine(OPD) is carried out and the plates are read with a spectrometer at 492nm.

Results. The histogram in FIG. 1 shows the expression of VCAM-1 on thehuman dermal cells stimulated for 24 hours with the various constructs.The results are expressed as an expression index, giving the activity ofhuman IFN-γ (500 U/mL: 75 ng/mL) a value of 1.

A marked induction of VCAM-1 is noted on the cells treated with thevectorized agonist (i.e. the MuL lipopeptide). This induction isdose-dependent: specifically, a differential expression of VCAM-1 isobserved depending on whether the cells were treated with 25 or 50 μM ofpeptide. The effect of the control construct (MuSL) can be explained bythe production of certain inflammatory cytokines such as TNF by thesecells in response to a state of stress due to the presence of thelipopeptide at high concentration. However, a marked difference instimulation is observed between MuL and MuSL. These results establishthe cytoplasmic addressing of the vectorized agonist, which conservesits biological activity, as confirmed by the observed induction ofVCAM-1. Furthermore, they establish that MuL is capable of stimulatingcells in a heterologous system, confirming a certain level of potentialof the vectorized agonist for use in a human system.

5-Induction of the Class II CMH Molecule (HLA-DR) on a Human Cell Line.

Principle: human colon carcinoma cells (COLO 205) are analysed for theirability to express HLA-DR after having been stimulated with the variouspeptide constructs.

Procedure. The cell line COLO 205 is cultured at a rate of 3×10⁵ cellsper well in a 24-well culture plate (NUNC). The following day, the cellsare stimulated with the various peptide constructs at a finalconcentration of 50 μM in 1 ml of medium, and are incubated at 37° C.for 24 hours. The cells are then recovered and labelled using 3 μg of ananti HLA-DR mouse biotinylated monoclonal antibody (clone L243). Afterrevelation with FITC streptavidin, the cells are analysed by flowcytometry.

Results.

Table 11 below shows the percentage of expression of HLA-DR quantifiedby flow cytometry on COLO 205 cells treated or not treated with 50 μM ofMuL or of MuSL.

TABLE 11 Untreated cells MuL MuSL Mu % HLA-DR 0.9 46.1 8.9 1.2

A marked induction of the expression of HLA-DR is observed on the COLO205 cells stimulated with MuL; this result confirms on a second modelthe immunostimulatory potential of MuL in a heterologous system. Thecontrol scrambled lipopeptide and the reference peptide described bySzente 5mu) [sic] have no activity in this model.

6 Induction of HLA-DR, ICAM-1 and VCAM-1 on Human Peripheral BloodMononuclear Cells (PBMC).

Principle. In the light of the results obtained on the human cell lineand on human primary cultures, the immunostimulatory potential of MuLwas evaluated on human cells taken directly from a healthy donor.

Procedure. PBMCs are isolated from a bag of blood and the cells are thencultured and stimulated for 24 hours with 25 and 50 μM of MuSL or MuL ina final volume of 1 ml. The cells are then recovered and labelled forthe expression of VCAM-1, ICAM-1 and HLA-DR according to the protocolalready described above. Finally, the cells are analysed by flowcytometry.

Results: table 12 below shows the percentage of VCAM-1, ICAM-1 andHLA-DR detected on PBMCs stimulated for 24 hours with MuSL and MuL.

TABLE 12 Untreated MuL MuSL cells 25 μM 50 μM 25 μM 50 μM HLA-DR 24.298.9 58.3 VCAM-1 17.9 62.6 97.1 39.7 86.9 ICAM-1 16.9 44 67.9 29.4 32

The results obtained on the PBMCs establish that MuL can induce, in adose-dependent manner, the expression of VCAM-1, ICAM-1 and HLA-DR oncells freshly taken from a patient. Despite an effect observed withMuSL, which might be due to the lipopeptide itself, a marked inductionof the various surface labels studied is observed on the cells treatedwith MuL. These results indicate that it is possible to use MuL in man.

7. Antiviral Effect

Principle: after having established in a human and a murine system thatMuL was capable of stimulating various cell types and of inducing theexpression of various surface labels, the ability of MuL to activate acell function, reflected by an effector function, was evaluated. To dothis, we studied the induction of an antiviral state on cells infectedwith the VSV virus. The induction of the antiviral state corresponds tothe total use of all the transduction pathways activated by the naturalcytokine. This test constitutes the standard test for assaying theactivity of recombinant IFN-γ batches.

Procedure.

L929 cells (mouse fibroblasts) are stimulated for 6 hours with murineIFN-γ, MuSL or MuL at different concentrations. After incubation for 24hours, VSV is added and the cells are incubated at 37° C. for 24 hours.The cells lysed with the virus are then removed by washing and the livecells are stained using a vital stain, a 1% solution of crystal violet.The staining with crystal violet is finally quantified by reading on aspectrophotometer at 570 nm.

Results. The histogram represented in FIG. 2 shows the results of theantiviral test carried out on L929 fibroblasts treated with the MuSL orMuL peptides. The live cells are stained with crystal violet and thisstaining is quantified by reading on a spectrophotometer at 570 nm. Theblack bars represent MuL and the white bars represent MuSL. Thehistogram represented in FIG. 3 shows the results of the antiviral testcarried out on L929 fibroblasts treated with recombinant IFN-γ.

The results obtained show a resistance to viral lysis in the cellstreated with MuL, whereas the MuSL control construct has no effect onviral lysis. This is noteworthy at very low concentrations of peptidesand thus establishes that MuL is capable of inducing an antiviral stateon cells. Furthermore, this test makes it possible to assay thebiological activity of our product with reference to the naturalcytokine. This test makes it possible to establish that 1 IU of IFN-γ iscontained in 5 to 6 μM of peptide, i.e. 2 to 3 μg of product. Given thatthis activation of an antiviral state is characteristic of IFN-γ, it maythus be concluded therefrom that MuL is capable of reproducing theeffects of the cytokine.

III. Conclusion

These studies establish that the peptide derived from the C-terminaldomain of IFN-γ modified with a palmitic acid is capable in all respectsof mimicking the effect of the cytokine, irrespective of the type ofcell used. It was thus established that its action takes place via aninteraction with the cytokine receptor. Furthermore, the resultsobtained in a heterologous system (human cells) and more particularly oncells freshly taken from patients confirm the fundamental value of theMuL construct. Specifically, this construct, which is found to be apowerful immunostimulator, which potentiates the antigenic presentation,which activates cell effector functions both on mouse cells and on humancells, can have a certain degree of therapeutic value, both via animmunostimulatory action and via an immunoadjuvant effect.

B) Immunomodulatory Potential of the MuL Lipopeptide Agonist of IFN-γ I.Effect on the Activity of IL-4

It is commonly described that the immunomodulatory potential of IFN-γlies in its ability to polarize the establishment of a type-1 T helper(Th1) response, while at the same time inhibiting the development of atype-2 T helper (Th2) response. This inhibitory action on thepolarization of the Th2 response takes place mainly via inhibition ofthe activity of IL-4. Thus, in order to show that the IFN-γ syntheticagonist is capable of inhibiting the biological effect of IL-4 onsplenocytes taken from mice, we used the following cell system. Thestimulation of splenocytes with an anti-CD40 antibody (antibody directedagainst a surface cell label: CD40) and IL-4 induces a proliferation ofthe murine B cells, following a synergistic action between the CD40 andIL-4 stimulation (Hasbold et al. 1994).

1. Procedure

The splenocytes taken from Balb/c mice are cultured and stimulated withanti-CD40 (10 μg/ml) and IL-4 (10 U/ml). 10 μM of the synthetic agonistof IFN-γ (MuL) or of its control lipopeptide (MuSL) are added. Afterstimulation for 24 hours, tritiated thymidine is added at a rate of 0.5μCurie per well. After incubation for 18 hours, the cells are filteredoff and the incorporation of tritiated thymidine is evaluated. Thisincorporation is proportional to the cell proliferation.

2. Results

The stimulation of the murine splenocytes with anti-CD40 (Ac CD40)results in a proliferation of the cells (FIG. 4). When IL-4 is added,there is a synergistic action of this cytokine and of the anti-CD40 onthe cell proliferation. This proliferation is specific to the anti-CD40and the IL-4, since IL-4 plus a control isotype antibody (Iso), have noeffect on the cell proliferation.

The addition of 10 μM of MuL results in an inhibition of the synergisticeffect of the anti-CD40 and the IL-4 on the proliferation of murinesplenocytes. The level of proliferation in the presence of MuL isidentical to that of the cells stimulated with the anti-CD40 alone. TheCD40+IL-4 synergistic effect is thus completely abolished. This isspecific to the IFN-γ agonist, since the addition of the controllipopeptide (MuSL) has no effect on the CD40/IL-4-dependentproliferation.

Thus, this experiment establishes that the synthetic agonist of IFN-γinhibits the biological activity of murine IL-4.

In order to demonstrate that the inhibition of the activity of IL-4 withMuL is specific to an IFN-γ agonist activity, it is thus establishedthat this inhibitory activity of MuL is restricted to cells whichexpress a functional IFN-γ receptor.

Thus, similar experiments were reproduced on cells taken from mice whichare deficient for the α chain of the IFN-γ receptor (IFN-γR KO) and onmice of the same genetic background and expressing a functional IFN-γreceptor (WT mice).

The results in FIGS. 5 and 6 demonstrate that the inhibition of thebiological activity of IL-4 with MuL is specific to an “IFN-γ like”activity, and that this activity takes place via the cytokine receptor.

The ability of the synthetic agonist to modulate the biological effectsof IL-4 suggests that MuL can be used in vivo, in particular in modelsof pulmonary hypersensitivity, given the prevalence of type-2 cytokinesin such pathologies. Specifically, by way of example, it has been shownin mice that the intranasal administration of recombinant IFN-γ inhibitsthe development of a pulmonary allergic response (Lack et al., 1996).Furthermore, the ability of the IFN-γ agonist to inhibit the activity ofIL-4 should facilitate, in vivo, the establishment of an immune responseof Th1 type, which suggests that this synthetic construct can be used invivo, as an immunomodulator, allowing preferential polarization of a Th1immune response.

II. Effect on the Synthesis of Immunoglobulins

In order to study in vitro the effect of MuL on the synthesis ofimmunoglobulins and to be able to assess the effects of the agonist onthe establishment of a humoral response in vivo, murine splenocytes withanti-CD40 were stimulated in vitro in the presence or absence of MuL.

1. Procedure

The splenocytes are stimulated under the following conditions:

unstimulated cells (0)

cells stimulated with 10 μg/ml of anti-CD40 (aCD40)

cells stimulated with 10 μg/ml of anti-CD40+MuL 10 μM (aCD40+MuL)

cells stimulated with 10 μg/ml of anti-CD40+MuSL 10 μM (aCD40+MuSL)

cells stimulated with the isotypic control of anti-CD40 (Iso)

cells stimulated with the isotypic control of anti-CD40+MuL 10 μM(Iso+MuL)

cells stimulated with the isotypic control of anti-CD40+MuSL 10 μM(Iso+MuSL).

2. Results

The results shown in FIGS. 7, 8 and 9 show that MuL potentiates theproduction of total IgG2a, IgG1 and IgG by splenocytes stimulated withan anti-CD40. This effect of MuL on the production of immunoglobulinsmay be the consequence of a helper effect of the agonist which isthought to stimulate the production of cytokines which would themselvespromote the synthesis of immunoglobulins.

These results show that MuL can in vivo, inter alia, contribute towardsthe establishment of the humoral response, and potentiate the immunityfollowing the injection of certain antigens which induce a humoralresponse.

III. Ex Vivo and In Vivo Study

In order to study the capacity of the MuL synthetic agonist of IFN-γ tostimulate the production of antibodies in vivo, the following protocolwas selected.

1. Procedure

Mice which are transgenic for the T cell receptor, specificallyrecognizing the Ova peptide (323-339) (Murphy et al., 1990) derived fromchicken ovalbumin, are immunized under the following conditions:

1—subcutaneous injection of 50 μg of Ova peptide (Ova)

2—subcutaneous injection of 50 μg of Ova peptide+50 μg of MuL (MuL+Ova)

3—subcutaneous injection of 50 μg of Ova peptide+50 μg of MuSL(MuSL+Ova)

4—subcutaneous injection, 24 hours before the immunization, of 50 μg ofMuL, followed by immunization the next day with 50 μg of Ova peptide(MuL 24 H and then Ova)

5—subcutaneous injection, 24 hours before the immunization, of 50 μg ofMuSL, followed by immunization the next day with 50 μg of Ova peptide(MuSL 24 H and then Ova)

The animals are sacrificed 15 days after the immunization. Theirsplenocytes are cultured and re-stimulated in vitro with 10 μg/ml ofanti-CD40 (1, 2, 3, 4, 5 and then aCD40) in the presence or absence ofMuL (1, 2, 3, 4, 5 and then aCD40+MuL) or of MuSL (1, 2, 3, 4, 5 andthen aCD40+MuSL) in order to be able to study the synthesis ofimmunoglobulins. The immunoglobulin isotypes are determined by aconventional ELISA technique.

2. Results

The comparative study of the immunization with Ova and MuL and Ova+MuSLshows a potentiation of the production of IgG2a following an aCD40+MuLre-stimulation, of cells taken from animals immunized with Ova+MuL.These results suggest that under conditions of secondary immunization,there is an increase in the production of IgG2a in these animals. Giventhat this antibody isotype is associated with a type-1 response in themouse, it may be assumed that MuL has allowed a preferentialpolarization of the immune response towards a Th1 response. It is alsoimportant to note that the injection of MuL 24 hours before immunizationwith Ova potentiates the synthesis of Ig2A. It thus appears that thesynthetic agonist has allowed a preferential initiation of the immuneresponse, which may explain the synthesis of IgG2a and the Th1polarization.

In contrast with the previous results (FIG. 10), the immunization withMuL+Ova does not potentiate the synthesis of IgG1. This confirms thepolarisation of the immune response observed in the experiment describedin FIG. 10. Specifically, it is commonly described that IFN-γ acts onthe humoral response by promoting the synthesis of IgG2a to thedetriment of that of IgG1.

The results shown in FIGS. 10 and 11 thus confirm a polarization of theimmune response towards a Th1 profile. This polarizing effect on theimmune response may be observed with other antigens associated withadjuvants or otherwise.

C) Study of Lipopeptides Consisting of Fragments of the Mu Peptide i.e.of the Lipopeptides L-mIFNγ 113-132 and L-mIFNγ 122-132

Since the relatively large size of the MuL compound described above(5000 Da) is liable to limit the efficacy of its passage across themembrane, the reduction in the length of the lipopeptides sequence whileat the same time preserving its activity to induce the expression ofclass II CMH molecules was studied on human and murine cells. Novellipopeptides derived from IFN-γ, as well as the study of the role of thelipid chain and the determination of the minimum active compound aredescribed below, in comparison with the results obtained with theabove-mentioned MuL and MuSL peptides.

1) Experimental Section

a) Peptide Synthesis and Characterization

The lipopeptide L-mIFNγ 113-132 corresponds to the peptide SEQ ID NO 1,i.e. to the fragment delimited by the amino acids located in positions113 and 132 of murine IFNγ, the C-terminal portion of which has beenmodified at the carboxamide end, the said peptide being linked via itsN-terminal end to an N^(α)-acetyl-lysine N^(ε)(palmitoyl) group (alsoreferred to as Ac-K(Pam) or L).

The lipopeptide L-mIFNγ 122-132 corresponds to the peptide SEQ ID NO 4,i.e. to the fragment delimited by the amino acids located in positions122 and 132 of murine IFNγ, the C-terminal portion of which has beenmodified at the carboxamide end, the said peptide being linked via itsN-terminal activity to an Ac-K(Pam) group.

The peptides and lipopeptides were synthesized on a Rink amide resin(Senn Chemicals A. G., Dielsdorf, CH) using the Fmoc-tBu strategy(Fields, G. B. and Noble, R. L., 1990; Merrifield, R. B., 1986), and byactivation with 2-(1H-benzothiazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HBTU)-hydroxybenzotriazole (HOBt) 0.45 M inN-methylpyrrolidinone (NMP). A systematic double coupling was carriedout using 4 equivalents of protected amino acids, followed by a step ofsystematic acetylation using acetic anhydride/DIPEA in NMP in thepresence of 0.5 M HOBt in an Applied Biosystem 430 A peptide synthesizer(Foster City, USA). An Fmoc Lys(Pam)-OH group (BACHEM, Bubendorf, CH)was incorporated into the N-terminal end in order to obtain thelipopeptides.

The peptides and lipopeptides were deprotected and removed from theresin by a treatment with trifluoroacetic acid (TFA) in the presence ofphenol/ethanedithiol/thioanisole/H₂O (0.75 g:250 μl:250 μl:500 μl). Thepeptides were isolated from the TFA solution by precipitation withdiethyl ether and lyophilized. Purifications were carried out by severalparallel passages on RP-HPLC in a 12.5 mm×250 mm column filled withNucleosil C18 (0.03, 5 μm) as stationary phase, using anacetonitrile/H₂O/0.05% TFA solvent system. The homogeneity was confirmedin two different RP-HPLC systems : all the peptides and lipopeptides aremore than 90% pure. Their identity was confirmed by determining theamino acid composition after total acid hydrolysis and by determiningthe molecular mass by TOF-PDMS (Bio-Ion 20 Plasma Desorption MassSpectrometer, Uppsala, Sweden). All the compounds are soluble in water.

The antepenultimate lysine 94 was introduced with a 4-methyltrityl (Mtt)protecting group for the synthesis of the fluorescent analogue ofL-mIFNγ 95-132 (MuL). After selective deprotection of the Mtt group with1% TFA in dichloromethane, the introduction onto the resin of5(6)-carboxytetramethylrhodamine was carried out by HBTU/HUBtactivation. After final deprotection with TFA and cleavage from theresin as described above, the fluorescent analogue was purified byprecipitation with diethyl ether.

b) Circular Dichroism Studies

The circular dichroism measurements on the peptides were carried out at25° C. using a Jobin-Yvon CD-6 machine at controlled temperature. Thescans were carried out with a cell 0.1 cm in length in an average timeof 5 sec. The wavelength interval measured ranges from 185 to 260 nm ata scanning step speed of 0.5 nm/step. The scans were carried out on thepeptides at neutral pH in a 2 mM phosphate buffer with or without thehelix-stabilizing reagent trifluoroethanol (TFE). The peptideconcentration was adjusted to a concentration of 20 μM, afterdetermination of the exact amount of a 100 μM solution by quantitativeamino acid analysis. The average values of 4 repeat scans were expressedas average molar ellipticity per residue (deg. cm² dmol.).

c) Cell Culturing and Stimulation

Fresh splenocytes are obtained from 7-week-old female 129 Sv mice. TheCOLO 205 human colon carcinoma lines are obtained from the ATCC(American Type Culture Collection). The splenocytes and COLO 205 cellsare maintained in RPMI 1640 (Gibco BRL, Courbevoie, France),supplemented with 10% FCS (Gibco BRL), sodium pyruvate (Sigma, St.Louis, USA) and incubated at 37° C. in 5% CO₂.

The cells were stimulated for 24 hours with different concentrations ofMuL, L-mIFNγ 113-132, L-mIFNγ 122-132 or MuSL. The murine splenocytesand COLO 205 cells were labelled for the expression of II MHC with 1 μgof anti-mouse IA^(b) FITC monoclonal antibody (mAb) (Pharmingen, SanDiego, USA) and 10 μl of anti-HLA-DR clone TAL.1B5 mouse FITC mAb(Cymbus Biotechnology Ltd, Hants, UK), respectively. A negative controlmouse FITC IgG1 (DAKO S. A., Trappes, France) was used as controlisotype. The cells were incubated for 1 hour at 4° C. in PBS, 10% FCSand then washed, and the expression of II MHC was analysed by flowcytometry using a Coulter EPILS II cytometer (Coulter, Hialeah, Fla.,USA) at a rate of 10,000 events per sample.

d) Immunolabelling and Fluorescence Microscopy

The intracellular passage of the lipopeptides was demonstrated in 1-10⁶splenocytes freshly obtained from mice, incubated for 10 minutes at 37°C. or 4° C. with 1 μM of rhodamine-labelled MuL lipopeptide. The cellswere washed twice with cold PBS and fixed with 4% paraformaldehyde inPBS for 15 minutes at 4° C. The fixed cells were permeabilized with0.05% NP-40, 1% BSA in PBS for 10 minutes at 4° C. and the non-specificsites were blocked with 2% BSA in PBS. Next, the cells were incubatedwith rabbit IgGs directed against the cytoplasmic domain of the α chainof IFNγ R (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif., USA).This antibody was used at a dilution of 1: 100. Fluorescein-coupledsheep anti-rabbit IgGs (Santa Cruz Biotechnology, Inc.) were used assecondary antibodies at a dilution of 1: 200. The cells were then washed4 times with PBS, mounted on slides and photographed in a Leica confocalmicroscope.

2) Results

All the peptides (indicated in Table 13 below) were obtained byautomated solid-phase synthesis and readily purified by RP-HPLC, exceptfor the rhodamine-labelled MuL analogue. The intercellular distributionof this fluorescent analogue was observed in the murine splenocytesafter incubation for 10 min. The 38-amino-acid lipopeptide was observedin the form of aggregates in close proximity to the plasma membrane, ina distribution compatible with the localization observed by its targetreceptor.

TABLE 13 Physiochemical characterizations of the lipopeptides studiedM.W. k’ k’ Peptide calculated found C3 C18 SEQ ID NO 22 Mu 4614 461320.81 9.46 SEQ ID NO 20 MuL 4981 4983 26.17 12.68 SEQ ID NO 21 L-mIFN-γ113-132 2824 2825 21.57 910.88 SEQ ID NO 25 L-mIFN-γ 122-132 1768 176919.85 10.53 SEQ ID NO 24 MuSL 4981 4979 22.59 11.24

The molecular weights (M. W.) of the peptides were determined by massspectrometry. The peptides were analysed by RP-HPLC in two differentsystems using either a Vydac C18 column (0.01-5 μm) (250×4.6 mm) or aZorbax C3 column (0.03-5 μm) (150×4.6 mm), eluted at 50° C. with anelution rate of 1 ml/min. Composition of the solvent: A=0.05% TFA inH₂O, B=0.05% TFA in H₂O/acetonitrile (20:80), at 215 nm, using a 0-100%B linear gradient over more than 60 min. The capacity factors k′C3 andk′C. 18 were measured in the two systems.

The mechanism involved in the transmembrane transfer is passive, ascould be observed after incubating the cells at 4° C., and is fastenough to avoid the complete degradation of the peptide by theexopeptidases in the culture medium.

The helical organization and the polycationic tail have been describedas being essential elements for the binding of an IFN-γ agonist peptideto its receptor (Szente et al., 1996). The peptide 108-132 has beendescribed as being the smallest peptide capable of binding to thecytoplasmic domain of IFN-γ R (Szente et al., 1996). Its capacity toinduce the expression of class II CMH molecules on murine cells wasreduced by a factor of 2 relative to the peptide 95-133 (Szente et al.,1996).

The crystalline structure of the homologous human cytokine (Ealick etal., 1991) shows the presence of 5 F helix turns in this portion of themolecule, corresponding to 18 of the 38 residues (47%) of biologicallyactive C-terminal peptide. Large portions of the MuL compound have beenamputated: the first 19 residues, including 3 of the 5 F helix turns,have been suppressed in the L-mIFN-γ 113-132 construct, and only 11residues of the C-terminal portion are present in L-mIFN-γ 122-132. Thecysteine found in position 133 (C-terminal end) of the cytokine has beenomitted in all the lipopeptides in order to avoid their dimerization byformation of disulphide, and replaced with a simple carboxamide end inorder to reinforce their stability with respect to carboxypeptidases.

The various lipopeptides were compared on the basis of their ability toinduce the expression of class II CMH molecules by murine splenocytes orhuman COLO 205 cell lines, incubated for 24 hours with differentconcentrations of lipopeptides (the non-lipid Mu peptide is inactiveunder these experimental conditions) (FIG. 12). A marked dose-dependentincrease in the class II CMH molecules was observed on the two celltypes by stimulation with MuL, or L-mIFN-γ 113-132, whereas neitherL-mIFN-γ 122-132 nor MuSL are active. This result rules out thepossibility of a non-specific induction of the expression of the classII CMH molecules by palmitic acid. The results indicated in FIG. 12 showthat the biological activity of the truncated lipopeptide L-mIFN-γ113-132 is equivalent to that of MuL on the splenocytes freshly obtainedfrom mice, with a 13-fold increase in the expression of class II CMHmolecules by cells stimulated with each of the lipopeptides at aconcentration of 50 μM (140 or 230 μg/ml, respectively). L-mIFN-γ113-132 is less active than MuL on the human cells, with a 10-foldincrease in the expression of HLA-DR as opposed to 22-fold at aconcentration of 50 μM. However, in contrast with MuL, highconcentrations of truncated lipopeptide are not cytotoxic: aconcentration of 75 μM (210 μg/ml) of this peptide induces a 15-foldgreater expression of HLA-DR for the human cell lines, and increases bya factor of 18 the expression of IA^(b) for the murine cells when usedat a concentration of 100 μM (280 μg/ml).

In order to further characterize the influence of the lipidmodification, circular dichroism studies on the peptides andlipopeptides were carried out in order to probe the conformationalchanges induced in the peptide by the lipid tail. FIG. 13 shows the CDspectra for Mu and MuL, obtained in a 2 mM pH 7 phosphate buffer atambient temperature with or without the helix-stabilizing reagenttrifluoroethanol. In the aqueous buffer, a small positive ellipticity at190 nm, and two minima at 203 and 218, suggest that the peptide is inrapid equilibrium between a poorly populated helical stage and adominant conformation which is extended or in the form of a random coil.In the presence of trifluoroethanol, the spectrum changes to acharacteristic organization with a high population of α helix with amaximum at 190 nm, and two minima at 209 and 221 nm. At 25% or 50% (byvol.) of trifluoroethanol, the ordered conformation reaches 53% or 65%respectively (considering that the value [θ]₂₂₂ for 100% helix=33,000).It is interesting to note that the spectrum of MuL in the buffer can besuperimposed on the spectrum of Mu in 25% of trifluoroethanol. Theaddition of 25% or 50% of trifluoroethanol to the lipopeptide solutionincreases the helical organization from 65% to 72% respectively, i.e. inrelative proportions that are greater than the theoretical helicalcontent of the corresponding segment in the natural cytokine.

The CD spectra of the truncated and scrambled lipopeptides are indicatedin FIGS. 14A (solutions in a buffer) and 14B (with 25% oftrifluoroethanol): the populations with a low content of helix or of βsheet which are observed in the buffer change to approximately 50-60%helical conformation by addition of 25% of trifluoroethanol.Surprisingly, this was even observed with the 12-amino-acid lipopeptideL-mIFN-γ 122-132, despite the absence of helical organization of the endof the cytokine in its natural context.

The surprising ability of these relatively large, water-solublecompounds to cross the cell membrane passively may be linked to theirtendency to spontaneously adopt an α-helical organization in water. Ifthe liposome-lipopeptide interaction can be considered as a model forthe cell-lipopeptide interaction, it may then be assumed that there isan insertion of the lipopeptides at the surface of the cells and then,in the particular case of the lipopeptides of the invention, a rapidtranslocation of the functional cargo sequence inside the cells, andtheir subsequent recognition of their target receptors.

3) Conclusion

The results given above show that the lipid modification has at least atwofold role, which contributes towards the stabilization of the helicalorganization of the associated peptide (even in the case of a shortpeptide lacking a helix) and towards its distribution in the cytoplasm.Since the shortest peptide described as being capable of binding IFN-γ Rand having a weak biological activity was the peptide 108-132 (Szente etal., 1996), the maintenance of the biological activity of a peptide 5residues shorter suggests that the lipid tail has another role which maycontribute towards stabilizing the peptide-receptor binding by means ofadditional hydrophobic interactions.

This study illustrates the apparent capacity of functional peptides toselectively recognize the biologically significant sites of vitalproteins, this being a property which is the basis of the development oflarge libraries of peptides as sources for the identification of ligandsfor various targets.

In this study, the introduction of a lipid tail improved the biologicalactivity of the basic peptide sequence, and its ability to reach itsintracellular receptor. The biological activity presented by theselipopeptide constructs derived from INF-γ confirms their use asimmunomodulators. Their biologically active concentration (about a fewhundred μg per ml) and their solubility in water (greater than 5 mg/ml)are compatible with volumes that are acceptable for injecting them.

Another advantage of the lipopeptides of the invention over recombinantcytokine is their good storage qualities, even in the event of aninterruption of the refrigeration conditions.

BIBLIOGRAPHIC REFERENCES

Alexander, J. et al., Immunity, 1:9, 751-761 (1994)

Ben Mohamed L., et al., Eur J Immunol, 27:5, 1242-1253 (1997)

Ealick S. E., Cook W. J., Vijay-Kumar S., Carson M., Nagabhusan T. L.,Trocta P. P., Bugg C. E (1991). Three-dimensional structure of the humanrecombinant interferon-γ. Science, 252:698-702

Estaquier J., et al., Molecular Immunology, 29:4, 489-499 (1992)

Fidler, I. J., Fogler, W. E., Kleinerman, E. S., Saiki, I. (1985).Abrogation of species specificity for activation of tumorocidalproperties in macrophages by recombinant mouse or human interferon-γencapsulated in liposomes. Journal of Immunology: 135:1289-1296.

Fields G. B., Noble R. L. (1990) Solid phase peptide synthesis utilizing9-fluorenylmethoxy carbonyl amino acids. Int. J Pept. Prot. Res. 35,161-214

Hasbold J, Johnson-Leger C, Atkins C J, Clark E A, Klaus GGB. 1994.Properties of mouse CD40: cellular distribution of CD40 and B cellactivation by monoclonal anti-mouse CD40 antibodies. Eur. J. Immunol. 24;1835-1842.

Krieger J. I., et al., J Immunol, 146:7, 2331-40 (1991)

Lack G, Bradley K L, Hamelmann E, Renz H, Loader J, Leung D Y M, LarsenG, Gelfand E W. 1996. Nebulized IFN-≢5 inhibits the development ofsecondary allergic response in mice. J. Immunol. 157:1432-1439.

E. Loing, A. Delanoye, C. Sergheraert, A. Tartar, H. Gras-Masse.Assessing delivery of lipopeptides into the cytoplasm of intact cells bya functional assay based on PKC inhibition. I. The Jurkat model. PeptideResearch., 9, 5, 229-232 (1996)

Merrifield, R. B; J Am Soc, 85, 2149-2154 (1963)

Merrifield, R. B; Science, 232, 341-347 (1986)

Murphy K M, Heimberger A B, Loh D Y. 1990. Induction by antigen ofintrathymic apoptosis of CD4⁺ CD8⁺ TCR¹⁰ thymocytes in vivo. Science250;1720-1723.

Panina-Bordignon P., et al., Eur J Immunol, 19:12, 2237-2242 (1989)

Sanceau, J., Sondermeyer, P., Beranger, F., Falcoff, R., Vaquero, C.(1I987). Intracellular human g—interferon triggers an antiviral state intransformed murine L cells. Proceedings of the National Academy ofsciences of USA. 84:2906-2910.

Sareneva T; Pirhonen J; Cantell K; Julkunen I N-glycosylation of humaninterferon-gamma:glycans at Asn-25 are critical for protease resistance.Biochem J 308 (Pt 1): 9-14 (1995)

Smith M R; Muegge K; Keller J R; Kung H F; Young H A; Durum S K Directevidence for an intracellular role for IFN-gamma. Microinjection ofhuman IFN-gamma induces Ia expression on murine macrophages. J Immunol144 :1777-82 (1990)

Szente B E; Johnson H M ; Binding of IFN gamma and its C-terminalpeptide to a cytoplasmic domain of its receptor that is essential forfunction. Biochem Biophys Res Commun 201:215-21 (1994)

Szente B E; Soos J M; Johnson H M; The C-terminus of IFN-gamma issufficient for intracellular function. Biochem Biophys Res Commun203:1645-54 (1994)

Szente B E; Subramaniam P S; Johnson H M; Identification of IFN-gammareceptor binding sites for JAK2 and enhancement of binding by IFN-gammaand its C-terminal peptide IFN-gamma (95-133). J Immunol 155:5617-22(1995)

Szente B E; Weiner I J; Jablonsky M J; Krishna N R; Torres B A; JohnsonH M (Department of Microbiology and Cell Science, University of Florida,Gainesville 32611, USA.) Structural requirements for agonist activity ofa murine interferon-gamma peptide. J Interferon Cytokine Res 16 :813-7(1996)

Tam P. and Spetzler J. C., Biomedical Peptides, Proteins & NucleicAcids, 1, 123-132 (1995)

Thiam, K., Loing, E., Gilles, F., Verwaerde, C., Quatannens, B.,Sergheraert, C., Auriault, C., Gras-Masse, H. Induction of apoptosis byPKC-pseudosubstrate lipopeptides in several human cells. Letters InPeptide Sciences, 4, 1-6, 1997.

KEY TO THE FIGURES

FIG. 1: Histogram representing the induction of VCAM-1 on human dermalcells (HMVECd). The results are expressed as an expression index, givingthe activity of human IFN-γ (500 U/ml; 75 ng/ml) a value of 1. Thecolumns from left to right correspond respectively to the resultsobtained:

without treating the said cells,

by treating the cells with human IFN-γ at 500 U/ml,

by treating the cells with TNF at 10 ng/ml,

by treating the cells with a mixture of human IFN-γ at 500 U/ml and TNFat 10ng/ml,

by treating the cells with the MuSL peptide at 25 μM,

by treating the cells with the MuSL peptide at 50 μM,

by treating the cells with the MuL peptide at 25 μM,

by treating the cells with the MuL peptide at 50 μM.

FIG. 2: Histogram representing the results of the antiviral test carriedout on L929 fibroblasts treated with the MuSL or MuL peptides. Theresults are expressed as optical density (OD). The columns from left toright correspond respectively to the measurements carried out:

on cells not infected with VSV,

on cells infected with VSV and not treated with MuSL or MuL,

on cells infected with VSV and treated with MuSL (white columns) or MuL(black columns) at 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM,10 μM

FIG. 3: Histogram representing the results of the antiviral test carriedout on L929 fibroblasts treated with recombinant IFN-γ. The results areexpressed as optical density (OD). The columns from left to rightcorrespond respectively to the measurements carried out:

on cells not infected with VSV,

on cells infected with VSV and not treated with recombinant IFN-γ,

on cells infected with VSV and treated with recombinant IFN-γ at 0.18IU, 0.38 IU, 0.75 IU, 1.5 IU, 3.12 IU, 6.25 IU, 12.5 IU, 25 IU, 50 IU,100 IU, 200 IU.

FIG. 4: Proliferative effect of the anti-CD40+IL-4 stimulation on murinesplenocytes. Inhibition by MuL of the biological activity of murineIL-4.

FIG. 5: Proliferative effect of the anti-CD40+IL-4 stimulation on murinesplenocytes taken from IFN-γR KO animals. In the absence of a functionalIFN-γ receptor, no inhibitory activity of MuL is observed.

FIG. 6: Proliferative effect of the anti-CD40+IL-4 stimulation on murinesplenocytes taken from WT animals. The presence of a functional IFN-γreceptor is necessary for MuL to be able to inhibit the biologicalactivity of IL-4.

FIG. 7: Synthesis of IgG2a by murine splenocytes stimulated in vitro byanti-CD40, in the presence or absence of MuL.

FIG. 8: Synthesis of IgG1 by murine splenocytes stimulated in vitro byanti-CD40, in the presence or absence of MuL.

FIG. 9: Synthesis of total IgGs by murine splenocytes stimulated invitro by anti-CD40, in the presence or absence of MuL.

FIG. 10: Production of IgG2a by the splenocytes of animals immunizedaccording to the various conditions described in the procedure andre-stimulated in vitro under the conditions noted on the y-axis. Thequantification of the synthesis of IgG2a is carried out by ELISA.

FIG. 11: Production of IgG1 by the splenocytes of animals immunizedaccording to the various conditions described in the procedure andre-stimulated in vitro under the conditions noted on the y-axis. Thequantification of the synthesis of IgG1 is carried out by ELISA.

FIG. 12: Induction of class II CMH molecules by human or murine cellsstimulated with lipopeptides derived from IFN-γ. The murine splenocytes(A) and the COLO 205 human cell line (B) were incubated for 24 hourswith different concentrations of lipopeptides derived from IFN-γ. Thecells were labelled with a monoclonal antibody directed against theclass II CMH molecules, and analysed by flow cytometry. The ratiobetween the average fluorescence intensity of the cells treated with thelipopeptides and between the average fluorescence intensity of theuntreated cells is indicated on the y-axis. The lipopeptideconcentration is indicated in μM on the x-axis; MuL: solid circles; MuSL: hollow circles ; L-mIFN 113-132: triangles; L-mIFN 122-132: X.

FIG. 13: CD (circular dichroism) spectra of Mu and MuL at aconcentration of 20 μM in a 2 mM pH 7 phosphate buffer without PFE (mIFN95-132: X; L-IFN 95-132: diamonds), with 25% of TFE (mIFN 95-132:triangles); L-mIFN 95-132: large squares), or with 50% of TFE (mIFN95-132: circles; L-mIFN 95-132: small squares). Temperature: 298 K. Thevalues indicated on the y-axis correspond to Theta×10⁻³ (deg.cm².dmo⁻¹),and those indicated on the x-axis correspond to wavelengths (nm).

FIG. 14: CD spectra of L-mIFN 113-132, L-mIFN 122-132 and MuSL, at aconcentration of 20 μM in a 2 mM pH 7 phosphate buffer (FIG. 14A), or inthe presence of 25% of TFE (FIG. 14B); (L-mIFN 113-132: triangles;L-mIFN 122-132: X; MuSL: hollow circles). The values indicated on they-axis correspond to Theta×10⁻³ (deg.cm².dmo⁻¹), and those indicated onthe x-axis correspond to wavelengths (nm).

TABLE 1 epitopes of chronic myeloid leukeaemia Peptide Sequence Bindingto HLA  247-255 (SEQ ID NO 26) B44  488-496 (SEQ ID NO 27) B44  768-776(SEQ ID NO 28) B44  901-934 b2a2 (SEQ ID NO 29) B44  902-935 b2a2 (SEQID NO 30) B44  986-994 (SEQ ID NO 31) B44 1176-1184 (SEQ ID NO 32) B441252-1260 (SEQ ID NO 33) B44 1691-1699 (SEQ ID NO 34) B44  49-57 (SEQ IDNO 35) B8  580-588 (SEQ ID NO 36) B8  722-730 (SEQ ID NO 37) B8  786-794(SEQ ID NO 38) B8  886-893 (SEQ ID NO 39) 88  928-936 b3a2 (SEQ ID NO40) B8 1830-1838 (SEQ ID NO 41) B8 1975-1983 (SEQ ID NO 42) B8 1977-1984(SEQ ID NO 43) B8  252-260 (SEQ ID NO 44) B7  329-338 (SEQ ID NO 45) B7 693-701 (SEQ ID NO 46) B7 1058-1066 (SEQ ID NO 47) B7 1196-1205 (SEQ IDNO 48) B7 1560-1569 (SEQ ID NO 49) B7 1717-1725 (SEQ ID NO 50) B71878-1884 (SEQ ID NO 51) B7  36-44 (SEQ ID NO 52) B27  71-79 (SEQ ID NO53) B27  575-583 (SEQ ID NO 54) B27  834-842 (SEQ ID NO 55) B27  642-650(SEQ ID NO 56) A2  684-692 (SEQ ID NO 57) A2  708-716 (SEQ ID NO 58) A2 714-722 (SEQ ID NO 59) A2  817-825 (SEQ ID NO 60) A2  881-889 (SEQ IDNO 61) A2  908-917 (SEQ ID NO 62) A2  912-920 (SEQ ID NO 63) A21240-1248 (SEQ ID NO 64) A2 1903-1911 (SEQ ID NO 65) A2 1932-1940 (SEQID NO 66) A2  50-58 (SEQ ID NO 67) A1  223-231 (SEQ ID NO 68) A1 549-558 (SEQ ID NO 69) A3/A11  583-591 (SEQ ID NO 70) A3/A11  715-724(SEQ ID NO 71) A3/A11  916-923 (SEQ ID NO 72) A3/A11  920-928 b3a2 (SEQID NO 73) A3/A11  924-932 b3a2 (SEQ ID NO 74) A3/A11 1156-1165 (SEQ IDNO 75) A3/A11 1311-1320 (SEQ ID NO 76) A3/A11 1499-1509 (SEQ ID NO 77)A3/A11 1724-1734 (SEQ ID NO 78) A3/A11 1905-1914 (SEQ ID NO 79) A3/A111922-1930 (SEQ ID NO 80) A3/A11  924-936 b3a2 (SEQ ID NO 81) DR4

TABLE 2 epitopes of p53 epitopes of p53 binding to HLA-A1 (SEQ ID NO 82)(196-205) (SEQ ID NO 83) (226-234) epitopes of p53 binding to HLA-A2(SEQ ID NO 84) (25-35) (SEQ ID NO 85) (65-73) (SEQ ID NO 86) (SEQ ID NO87) (129-137) (SEQ ID NO 88) (149-157) (SEQ ID NO 89) (187-197) (SEQ IDNO 90) (264-272) (SEQ ID NO 91) (322-330) epitopes of p53 binding toHLA-A3 (SEQ ID NO 92) (156-164) (SEQ ID NO 93) (282-290) (SEQ ID NO 94)(298-306) epitopes of p53 binding to HLA-B7 (SEQ ID NO 95) (26-35) (SEQID NO 96) (63-73) (SEQ ID NO 97) (SEQ ID NO 98) (189-197) (SEQ ID NO 99)(249-257) (SEQ ID NO 100) (321-330) epitopes of p53 binding to HLA-B8(SEQ ID NO 101) (135-143) (SEQ ID NO 102) (187-195) (SEQ ID NO 103)(210-218) epitopes of p53 binding to HLA-B51 (SEQ ID NO 104) (25-35)(SEQ ID NO 105) (65-73) (SEQ ID NO 106) (194-203)

TABLE 3 epitopes of human melanoma Position of the Gene/protein MHCrestriction Peptide amino acids Tyrosinase HLA-A2 (SEQ ID NO 107) 1-9HLA-A2 (SEQ ID NO 108) 369-377 (SEQ ID NO 109) HLA-A24 (SFQ ID NO 110)206-214 HLA-B44 (SEQ ID NO 111) 192-200 HLA-DR4 (SEQ ID NO 112) 56-70(SEQ ID NO 113) 450-462 Pmel17^(gp100) HLA-A2 (SEQ ID NO 114) 154-162HLA-A2 (SEQ ID NO 115) 177-186 HLA-A2 (SEQ ID NO 116) 178-186 HLA-A2(SEQ ID NO 117) 209-217 HLA-2 (SEQ ID NO 118) 280-288 HLA-A2 (SEQ ID NO119) 457-466 (SEQ ID NO 120) HLA-A2 (SEQ ID NO 121) HLA-A2 (SEQ ID NO122) 570-579 HLA-A3 17-25 Melan-A^(MART-1) HLA-A2 (SEQ ID NO 123)26(7)-35  HLA-A2 (SEQ ID NO 124) 32-40 gp^(75TRP-1) HLA-A31 (SEQ ID NO125) TRP-2 HLA-A31 (SEQ ID NO 126) 197-205

TABLE 4 epitopes of tumours resulting from mutations MHC Position of theGene/protein Tumour restriction Peptide amino acids MUM-1 MelanomaHLA-B44 (SEQ ID NO 30-38 127) CDK4 Melanoma HLA-A2 (SEQ ID NO 23-32 128)β-catenin Melanoma HLA-A24 (SEQ ID NO 29-37 129) HLA-A2 renal carcinoma— — — CASP-8 squamous carcinoma of HLA-B35 (SEQ ID NO 476-484 the headand 130) neck

TABLE 5 antigens common to various tumours tissue in which Positionnormal of the expression MHC amino Gene takes place restrictionAntigenic peptide acids MAGE-1 testicles HLA-A1 (SEQ ID NO 131) 161-169HLA-Cw16 (SEQ ID NO 132) 230-238 MAGE-3 testicles HLA-A1 (SEQ ID NO 133)168-176 HLA-A2 (SEQ ID NO 134) 271-279 HLA-B44 (SEQ ID NO 135) 167-176BAGE testicles HLA-Cw16 (SEQ ID NO 136)  2-10 GAGE-1/2 testicles HLA-Cw6(SEQ ID NO 137)  9-16 RAGE-1 retina HLA-B7 (SEQ ID NO 138) 11-20 GnTVnone HLA-A2 (SEQ ID NO 139) 38-64 mucin breasts no (SEQ ID NO 140)*during restrictions lactation *aberrant N-acetyl glucosaminyltransferase V (GnTV) transcript found only in the melanomas.

TABLE 6 epitopes of the HIV-1 virus HLA-A1 (Nef 96-106: (SEQ ID NO 141)(Nef 121-128 (SEQ ID NO 142) (Nef 137-145: (SEQ ID NO 143) (Nef 184-191:(SEQ ID NO 144) (Nef 195-202: (SEQ ID NO 145) HLA-A2 Gp120 121-129: (SEQID NO 146) P17 77-85: (SEQ ID NO 147) RT 200-208: (SEQ ID NO 148) RT275-285: (SEQ ID NO 149) RT 346-354: (SEQ ID NO 150) RT 368-376: (SEQ IDNO 151) RT 376-387: (SEQ ID NO 152) RT 476-484: (SEQ ID NO 153) RT588-596: (SEQ ID NO 154) RT 683-692: (SEQ ID NO 155) Nef 136-145: (SEQID NO 156) Nef 180-189: (SEQ ID NO 157) Nef 190-198: (SEQ ID NO 158)Gp41 818-826: (SEQ ID NO 159) P24 185-193: (SEQ ID NO 160) RT 346-354:(SEQ ID NO 161) RT 588-596: (SEQ ID NO 162) Pro 143-152: (SEQ ID NO 163)(Gp 120 37-44: (SEQ ID NO 164) (Gp 120 115-122: (SEQ ID NO 165) (Gp 120313-321: (SEQ ID NO 166) (Gp 120 197-205: (SEQ ID NO 167) (Gp 120428-435: (SEQ ID NO 168) (Gp 41 836-844: (SEQ ID NO 169) (p24 219-228:(SEQ ID NO 170) (p15 422-431: (SEQ ID NO 171) (p15 448-456: (SEQ ID NO172) (RT 681-691: (SEQ ID NO 173) HLA-A3 P17 18-26: (SEQ ID NO 174) P1720-28: (SEQ ID NO 175) RT 200-210: (SEQ ID NO 176) RT 325-333: (SEQ IDNO 177) RT 359-368: (SEQ ID NO 178) Nef 73-82: (SEQ ID NO 179) Gp 12037-46: (SEQ ID NO 180) Gp41 775-785: (SEQ ID NO 181) P17 18-26: (SEQ IDNO 182) HLA-A11 RT 325-333: (SEQ ID NO 183) RT 507-517: (SEQ ID NO 184)Nef 73-82: (SEQ ID NO 185) Nef 84-92: (SEQ ID NO 186) p24 349-359: (SEQID NO 187) P17 83-91: (SEQ ID NO 188) HLA-A24 (A9) Gp120 52-61: (SEQ IDNO 189) Gp41 591-598: (SEQ ID NO 190) or 590-597: (SEQ ID NO 191) (RT484-492: (SEQ ID NO 192) (RT 508-516: (SEQ ID NO 193) (RT 681-691: (SEQID NO 194) HLA-A25 (A10) P24 203-212: (SEQ ID NO 195) HLA-A26 (A10) P24167-175: (SEQ ID NO 196) HLA-A30 (A19) (Gp41 845-852: (SEQ ID NO 197)HLA-A31 (A19) Gp41 775-785: (SEQ ID NO 198) HLA-A32 (A19) Gp120 424-432:(SEQ ID NO 199) Gp41 774-785: (SEQ ID NO 200) RT 559-568: (SEQ ID NO201) HLA-A33 (A19) (P24 266-275: (SEQ ID NO 202) HLA-B7 RT 699-707: (SEQID NO 203) Nef 68-77: (SEQ ID NO 204) Nef 128-137: (SEQ ID NO 205) Gp120303-312: (SEQ ID NO 206) Gp41 848-856: (SEQ ID NO 207) RT 699-707: (SEQID NO 208) HLA-B8 Gp120 2-10: (SEQ ID NO 209) P17 24-32: (SEQ ID NO 210)Nef 90-97: (SEQ ID NO 211) P24 259-267: (SEQ ID NO 212) Gp41 591-598:(SEQ ID NO 213) (Gp41 849-856: PRRIRQGL (SEQ ID NO 214) or 851-859:RIRQGLERIL (SEQ ID NO 215) (P24 329-337: (SEQ ID NO 216) (RT 185-193:(SEQ ID NO 217) (Nef 182-189: (SEQ ID NO 218) HLA-B14 Gp4l 589-597: (SEQID NO 219) P24 298-306: (SEQ ID NO 220) (P24 183-191 ?: (SEQ ID NO 221)(p24 304-313: (SEQ ID NO 222) (p24 305-313: (SEQ ID NO 223) HLA-B18 Nef135-143: (SEQ ID NO 224) Nef 135-143: (SEQ ID NO 225) HLA-B27 P24263-272: (SEQ ID NO 226) Nef 73-82: (SEQ ID NO 227) Nef 134-141: (SEQ IDNO 228) or 133-141: (SEQ ID NO 229) Gp41 589-597: (SEQ ID NO 230) (Gp41791-800: (SEQ ID NO 231) HLA-B35 Gp120 78-86: (SEQ ID NO 232) Gp120257-265: (SEQ ID NO 233) RT 285-294: (SEQ ID NO 234) RT 323-331: (SEQ IDNO 235) RT 342-350: (consensus clade B) (SEQ ID NO 236) RT 460-468: (SEQID NO 237) RT 598-608: (SEQ ID NO 238) Nef 68-76: (SEQ ID NO 239) Nef74-81: (SEQ ID NO 240) Gp41 611-619: (SEQ ID NO 241) Gp120 42-52: (SEQID NO 242) P17 124-132: (consensus clade B) (SEQ ID NO 243) P24 254-262:(consensus clade B) (SEQ ID NO 244) HLA-B37 Nef 120-128: (SEQ ID NO 245)HLA-B44 (B12) P24 178-186: (SEQ ID NO 246) (p24 175-184: (SEQ ID NO 247)HLA-B51 (B5) gp41 562-570: (SEQ ID NO 248) RT 200-208: (SEQ ID NO 249)RT 209-217: (SEQ ID NO 250) RT 295-302: (SEQ ID NO 251) HLA-B52 (B5) Nef190-198: (SEQ ID NO 252) HLA-B55 (B22) Gp120 42-51: (SEQ ID NO 253)HLA-B57 and B58 P24 240-249: (SEQ ID NO 254) (B17) Nef 116-125: (SEQ IDNO 255) or 116-124: (SEQ ID NO 256) Nef 120-128: (SEQ ID NO 257) (P24147-155: (SEQ ID NO 258) (P24 164-172: (SEQ ID NO 259) HLA-Bw62 (B15)P17 20-29: (SEQ ID NO 260) P24 268-277: (SEQ ID NO 261) RT 427-438: (SEQID NO 262) Nef 84-91: (SEQ ID NO 263) Nef 117-127: (SEQ ID NO 264)HLA-Cw4 gp120 380-388: (SEQ ID NO 265) HLA-Cw8 RT 663-672: (SEQ ID NO266) P24 305-313: (SEQ ID NO 267) Nef 82-91: (SEQ ID NO 268) HLA-Cw? P24308-316: (SEQ ID NO 269)

TABLE 7 epitopes of the E6 and E7 proteins (E7 11-20) (SEQ ID NO 270)(E7 82-90) (SEQ ID NO 271) (E7 86-93) (SEQ ID NO 272) (E6 29-38) (SEQ IDNO 273) (E6 18-26) (SEQ ID NO 274) (E6 8-15) (SEQ ID NO 275) (E6 45-67)(SEQ ID NO 276) (E6 80-88) (SEQ ID NO 277) (E6 121-140) (SEQ ID NO 278)(E7 43-57) (SEQ ID NO 279) (E7 44-52) (SEQ ID NO 280) (E7 46-55) (SEQ IDNO 281)

281 1 20 PRT Mus sp. 1 Ile Arg Val Val His Gln Leu Leu Pro Glu Ser SerLeu Arg Lys Arg 1 5 10 15 Lys Arg Ser Arg 20 2 143 PRT Homo sapiens 2Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn 1 5 1015 Ala Gly His Ser Asp Val Ala Asp Asn Gly Thr Leu Phe Leu Gly Ile 20 2530 Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln Ser Gln 35 4045 Ile Val Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gln 50 5560 Ser Ile Gln Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys 65 7075 80 Phe Phe Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr 8590 95 Asn Tyr Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu100 105 110 Leu Ile Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr GlyLys 115 120 125 Arg Lys Arg Ser Gln Met Leu Phe Arg Gly Arg Arg Ala SerGln 130 135 140 3 143 PRT Bovine sp. 3 Gln Gly Gln Phe Phe Arg Glu IleGlu Asn Leu Lys Glu Tyr Phe Asn 1 5 10 15 Ala Ser Ser Pro Asp Val AlaLys Gly Gly Pro Leu Phe Ser Glu Ile 20 25 30 Leu Lys Asn Trp Lys Asp GluSer Asp Lys Lys Ile Ile Gln Ser Gln 35 40 45 Ile Val Ser Phe Tyr Phe LysLeu Phe Glu Asn Leu Lys Asp Asn Gln 50 55 60 Val Ile Gln Arg Ser Met AspIle Ile Lys Gln Asp Met Phe Gln Lys 65 70 75 80 Phe Leu Asn Gly Ser SerGlu Lys Leu Glu Asp Phe Lys Lys Leu Ile 85 90 95 Gln Ile Pro Val Asp AspLeu Gln Ile Gln Arg Lys Ala Ile Asn Glu 100 105 110 Leu Ile Lys Val MetAsn Asp Leu Ser Pro Lys Ser Asn Leu Arg Lys 115 120 125 Arg Lys Arg SerGln Asn Leu Phe Arg Gly Arg Arg Ala Ser Met 130 135 140 4 143 PRTCallithrix jacchus 4 Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys LysTyr Phe Asn 1 5 10 15 Ala Gly Asp Ser Asp Val Ala Asp Asn Gly Thr LeuPhe Leu Asp Ile 20 25 30 Leu Arg Thr Trp Arg Glu Glu Gly Asp Arg Lys IleMet Gln Ser Gln 35 40 45 Ile Ile Ser Phe Tyr Phe Lys Leu Phe Lys Asn PheLys Asp Asn Gln 50 55 60 Ser Ile Gln Lys Ser Met Glu Thr Ile Lys Glu AspMet Asn Val Lys 65 70 75 80 Phe Phe Asn Ser Asn Lys Arg Lys Gln Asp AspPhe Glu Arg Leu Thr 85 90 95 Asn Tyr Ser Val Asn Asp Leu Asn Val Gln ArgLys Ala Ile His Glu 100 105 110 Leu Ile Gln Val Met Ala Glu Leu Ser ProAla Pro Lys Ile Gly Lys 115 120 125 Arg Arg Arg Ser Gln Thr Leu Phe ArgGly Arg Arg Ala Ser Gln 130 135 140 5 142 PRT Cercocebus torquatius 5Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn 1 5 1015 Ala Gly Asp Pro Asp Val Ala Asp Asn Gly Thr Leu Phe Leu Asp Ile 20 2530 Leu Arg Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln Ser Gln 35 4045 Ile Val Ser Phe Tyr Phe Lys Leu Phe Lys Ser Phe Lys Asp Asp Gln 50 5560 Arg Ile Gln Lys Ser Val Glu Thr Ile Lys Glu Asp Ile Asn Val Lys 65 7075 80 Phe Phe Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr 8590 95 Asn Tyr Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Val His Glu100 105 110 Leu Ile Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Ile GlyLys 115 120 125 Arg Lys Arg Ser Gln Thr Phe Arg Gly Arg Arg Ala Ser Gln130 135 140 6 145 PRT Canis familiaris 6 Tyr Cys Gln Ala Met Phe Phe LysGlu Ile Glu Asn Leu Lys Glu Tyr 1 5 10 15 Phe Asn Ala Ser Asn Pro AspVal Ser Asp Gly Gly Ser Leu Phe Val 20 25 30 Asp Ile Leu Lys Lys Trp ArgGlu Glu Ser Asp Lys Thr Ile Ile Gln 35 40 45 Ser Gln Ile Val Ser Phe TyrLeu Lys Leu Phe Asp Asn Phe Lys Asp 50 55 60 Asn Gln Ile Ile Gln Arg SerMet Asp Thr Ile Lys Glu Asp Met Leu 65 70 75 80 Gly Lys Phe Leu Asn SerSer Thr Ser Lys Arg Glu Asp Phe Leu Lys 85 90 95 Leu Ile Gln Ile Pro ValAsn Asp Leu Gln Val Gln Arg Lys Ala Ile 100 105 110 Asn Glu Leu Ile LysVal Met Asn Asp Leu Ser Pro Arg Ser Asn Leu 115 120 125 Arg Lys Arg LysArg Ser Gln Asn Leu Phe Arg Gly Arg Arg Ala Ser 130 135 140 Lys 145 7144 PRT Felis sp. 7 Gln Ala Met Phe Phe Lys Glu Ile Glu Glu Leu Lys GlyTyr Phe Asn 1 5 10 15 Ala Ser Asn Pro Asp Val Ala Asp Gly Gly Ser LeuPhe Val Asp Ile 20 25 30 Leu Lys Asn Trp Lys Glu Glu Ser Asp Lys Thr IleIle Gln Ser Gln 35 40 45 Ile Val Ser Phe Tyr Leu Lys Met Phe Glu Asn LeuLys Asp Asp Asp 50 55 60 Gln Arg Ile Gln Arg Ser Met Asp Thr Ile Lys GluAsp Met Leu Asp 65 70 75 80 Lys Leu Leu Asn Thr Ser Ser Ser Lys Arg AspAsp Phe Leu Lys Leu 85 90 95 Ile Gln Ile Pro Val Asn Asp Leu Gln Val GlnArg Lys Ala Ile Asn 100 105 110 Glu Leu Phe Lys Val Met Asn Asp Leu SerPro Arg Ser Asn Leu Arg 115 120 125 Lys Arg Lys Arg Ser Gln Asn Leu PheArg Gly Arg Arg Ala Ser Lys 130 135 140 8 143 PRT Cervidae sp. 8 Gln GlyPro Phe Phe Lys Glu Ile Glu Asn Leu Lys Glu Tyr Phe Asn 1 5 10 15 AlaSer Asn Pro Asp Val Ala Glu Gly Gly Pro Leu Phe Ile Glu Ile 20 25 30 LeuLys Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile Ile Gln Ser Gln 35 40 45 IleVal Ser Phe Tyr Phe Lys Leu Phe Glu Asn Phe Lys Asp Asn Gln 50 55 60 ValIle Gln Arg Ser Val Asp Ile Ile Lys Gln Asp Met Phe Gln Lys 65 70 75 80Phe Leu Asn Gly Ser Ser Glu Lys Leu Glu Asp Phe Lys Lys Leu Ile 85 90 95Gln Ile Ser Val Asp Asp Met Gln Ile Gln Arg Lys Ala Ile Asn Glu 100 105110 Leu Ile Lys Val Met Asn Asp Leu Ser Pro Lys Ser Asn Leu Ile Lys 115120 125 Arg Lys Arg Ser Gln Asn Leu Phe Arg Gly Arg Arg Ala Ser Met 130135 140 9 140 PRT Gallus sp. 9 Leu Asn Leu Val Gln Leu Gln Asp Asp IleAsp Lys Leu Lys Ala Asp 1 5 10 15 Phe Asn Ser Ser His Ser Asp Val AlaAsp Gly Gly Pro Ile Ile Val 20 25 30 Glu Lys Leu Lys Asn Trp Thr Glu ArgAsn Glu Lys Arg Ile Ile Leu 35 40 45 Ser Gln Ile Val Ser Met Tyr Leu GluMet Leu Glu Asn Thr Asp Lys 50 55 60 Ser Lys Pro His Ile Lys His Ile SerGlu Glu Leu Tyr Thr Leu Lys 65 70 75 80 Asn Asn Leu Pro Asp Gly Val LysLys Val Lys Asp Ile Met Asp Leu 85 90 95 Ala Lys Leu Pro Met Asn Asp LeuArg Ile Gln Arg Lys Ala Ala Asn 100 105 110 Glu Leu Phe Ser Ile Leu GlnLys Leu Val Asp Pro Pro Ser Phe Lys 115 120 125 Arg Lys Arg Ser Gln SerGln Arg Arg Cys Asn Cys 130 135 140 10 143 PRT Equus caballus 10 Gln AlaAla Phe Phe Lys Glu Ile Glu Asn Leu Lys Glu Tyr Phe Asn 1 5 10 15 AlaSer Asn Pro Asp Val Gly Asp Gly Gly Pro Leu Phe Leu Asp Ile 20 25 30 LeuLys Asn Trp Lys Glu Asp Ser Asp Lys Lys Ile Ile Gln Ser Gln 35 40 45 IleVal Ser Phe Tyr Phe Lys Leu Phe Glu Asn Leu Lys Asp Asn Gln 50 55 60 ValIle Gln Lys Ser Met Asp Thr Ile Lys Glu Asp Leu Phe Val Lys 65 70 75 80Phe Phe Asn Ser Ser Thr Ser Lys Leu Glu Asp Phe Gln Lys Leu Ile 85 90 95Gln Ile Pro Val Asn Asp Leu Lys Val Gln Arg Lys Ala Ile Ser Glu 100 105110 Leu Ile Lys Val Met Asn Asp Leu Ser Pro Lys Ala Asn Leu Arg Lys 115120 125 Arg Lys Arg Ser Gln Asn Pro Phe Arg Gly Arg Arg Ala Leu Gln 130135 140 11 142 PRT Macaca sp. 11 Gln Asp Pro Tyr Val Lys Glu Ala Glu AsnLeu Lys Lys Tyr Phe Asn 1 5 10 15 Ala Gly Asp Pro Asp Val Ala Asp AsnGly Thr Leu Phe Leu Asp Ile 20 25 30 Leu Arg Asn Trp Lys Glu Glu Ser AspArg Lys Ile Met Gln Ser Gln 35 40 45 Ile Val Ser Phe Tyr Phe Lys Leu PheLys Asn Phe Lys Asp Asp Gln 50 55 60 Arg Ile Gln Lys Ser Val Glu Thr IleLys Glu Asp Ile Asn Val Lys 65 70 75 80 Phe Phe Asn Ser Asn Lys Lys LysArg Asp Asp Phe Glu Lys Leu Thr 85 90 95 Asn Tyr Ser Val Thr Asp Ser AsnVal Gln Arg Lys Ala Val His Glu 100 105 110 Leu Ile Gln Val Met Ala GluLeu Ser Pro Ala Ala Lys Ile Gly Lys 115 120 125 Arg Lys Arg Ser Gln MetPhe Arg Gly Arg Arg Ala Ser Gln 130 135 140 12 143 PRT Sus scrofa 12 GlnAla Pro Phe Phe Lys Glu Ile Thr Ile Leu Lys Asp Tyr Phe Asn 1 5 10 15Ala Ser Thr Ser Asp Val Pro Asn Gly Gly Pro Leu Phe Leu Glu Ile 20 25 30Leu Lys Asn Trp Lys Glu Glu Ser Asp Lys Lys Ile Ile Gln Ser Gln 35 40 45Ile Val Ser Phe Tyr Phe Lys Phe Phe Glu Ile Phe Lys Asp Asn Gln 50 55 60Ala Ile Gln Arg Ser Met Asp Val Ile Lys Gln Asp Met Phe Gln Arg 65 70 7580 Phe Leu Asn Gly Ser Ser Gly Lys Leu Asn Asp Phe Glu Lys Leu Ile 85 9095 Lys Ile Pro Val Asp Asn Leu Gln Ile Gln Arg Lys Ala Ile Ser Glu 100105 110 Leu Ile Lys Val Met Asn Asp Leu Ser Pro Arg Ser Asn Leu Arg Lys115 120 125 Arg Lys Arg Ser Gln Thr Met Phe Gln Gly Gln Arg Ala Ser Lys130 135 140 13 144 PRT Oryctolagus cuniculus 13 Gln Asp Thr Leu Thr ArgGlu Thr Glu His Leu Lys Ala Tyr Leu Lys 1 5 10 15 Ala Asn Thr Ser AspVal Ala Asn Gly Gly Pro Leu Phe Leu Asn Ile 20 25 30 Leu Arg Asn Trp LysGlu Glu Ser Asp Asn Lys Ile Ile Gln Ser Gln 35 40 45 Ile Val Ser Phe TyrPhe Lys Leu Phe Asp Asn Leu Lys Asp His Glu 50 55 60 Val Ile Lys Lys SerMet Glu Ser Ile Lys Glu Asp Ile Phe Val Lys 65 70 75 80 Phe Phe Asn SerAsn Leu Thr Lys Met Asp Asp Phe Gln Asn Leu Thr 85 90 95 Arg Ile Ser ValAsp Asp Arg Leu Val Gln Arg Lys Ala Val Ser Glu 100 105 110 Leu Ser AsnVal Leu Asn Phe Leu Ser Pro Lys Ser Asn Leu Lys Lys 115 120 125 Arg LysArg Ser Gln Thr Leu Phe Arg Gly Arg Arg Ala Ser Lys Tyr 130 135 140 14143 PRT Ovis sp. 14 Gln Gly Pro Phe Phe Lys Glu Ile Glu Asn Leu Lys GluTyr Phe Asn 1 5 10 15 Ala Ser Asn Pro Asp Val Ala Lys Gly Gly Pro LeuPhe Ser Glu Ile 20 25 30 Leu Lys Asn Trp Lys Glu Glu Ser Asp Lys Lys IleIle Gln Ser Gln 35 40 45 Ile Val Ser Phe Tyr Phe Lys Leu Phe Glu Asn LeuLys Asp Asn Gln 50 55 60 Val Ile Gln Arg Ser Met Asp Ile Ile Lys Gln AspMet Phe Gln Lys 65 70 75 80 Phe Leu Asn Gly Ser Ser Glu Lys Leu Glu AspPhe Lys Arg Leu Ile 85 90 95 Gln Ile Pro Val Asp Asp Leu Gln Ile Gln ArgLys Ala Ile Asn Glu 100 105 110 Leu Ile Lys Val Met Asn Asp Leu Ser ProLys Ser Asn Leu Arg Lys 115 120 125 Arg Lys Arg Ser Gln Asn Leu Phe ArgGly Arg Arg Ala Ser Met 130 135 140 15 143 PRT Mus montanus 15 Gln AspThr Val Asn Lys Glu Ile Glu Asp Leu Lys Gly Tyr Phe Asn 1 5 10 15 AlaSer Asn Ser Asn Val Ser Asp Gly Gly Ser Leu Phe Leu Asp Ile 20 25 30 LeuAsp Lys Trp Lys Glu Glu Ser Asp Lys Lys Val Ile Gln Ser Gln 35 40 45 ValVal Ser Phe Tyr Phe Lys Leu Phe Glu His Leu Lys Asp Asn Lys 50 55 60 AsnIle Gln Arg Ser Met Asp Thr Ile Lys Gly Asp Leu Phe Ala Lys 65 70 75 80Phe Phe Asn Ser Ser Thr Asn Lys Leu Gln Asp Phe Leu Lys Val Ser 85 90 95Gln Val Gln Val Asn Asp Leu Lys Ile Gln Arg Lys Ala Val Ser Glu 100 105110 Leu Lys Lys Val Met Asn Asp Leu Leu Pro His Ser Thr Leu Arg Lys 115120 125 Arg Lys Arg Ser Gln Ser Ser Ile Arg Gly Arg Arg Ala Ser Lys 130135 140 16 151 PRT Meriones unguiculatus 16 Gln Val Pro Ile Ile Glu GluIle Glu Asn Leu Lys Arg Tyr Phe Asn 1 5 10 15 Ser Ser Asn Ser Ala ValGly Asp Ser Lys Asp Val Val Leu His Val 20 25 30 Leu Arg Asn Trp Gln GluAsp Gly Asp Thr Lys Val Ile Asp Val Gln 35 40 45 Ile Val Ser Phe Tyr PheLys Leu Phe Glu Ala Leu Lys Gly Asn Gln 50 55 60 Ala Ile Glu Lys Ser IleAsn Ala Ile Arg Ala Asp Leu Ile Ala Asn 65 70 75 80 Phe Phe Asn Asn SerGlu Ala Lys Tyr Asp Gly Phe Met Ser Ile Met 85 90 95 Lys Ile Glu Val AsnAsp Pro Gln Ile Gln Ser Lys Ala Ile Asn Glu 100 105 110 Leu Val Lys ValMet Gly His Leu Ser Pro Arg Val Thr Leu Arg Lys 115 120 125 Arg Lys ArgSer Arg Cys Cys Phe Gly Gly Gly Asn Arg Leu Asn Lys 130 135 140 Asn AsnPro Ala Ser Thr Ile 145 150 17 133 PRT Mus sp. 17 His Gly Thr Val IleGlu Ser Leu Glu Ser Leu Asn Asn Tyr Phe Asn 1 5 10 15 Ser Ser Gly IleAsp Val Glu Glu Lys Ser Leu Phe Leu Asp Ile Trp 20 25 30 Arg Asn Trp GlnLys Asp Gly Asp Met Lys Ile Leu Gln Ser Gln Ile 35 40 45 Ile Ser Phe TyrLeu Arg Leu Phe Glu Val Leu Lys Asp Asn Gln Ala 50 55 60 Ile Ser Asn AsnIle Ser Val Ile Glu Ser His Leu Ile Thr Thr Phe 65 70 75 80 Phe Ser AsnSer Lys Ala Lys Lys Asp Ala Phe Met Ser Ile Ala Lys 85 90 95 Phe Glu ValAsn Asn Pro Gln Val Gln Arg Gln Ala Phe Asn Glu Leu 100 105 110 Ile ArgVal Val His Gln Leu Leu Pro Glu Ser Ser Leu Arg Lys Arg 115 120 125 LysArg Ser Arg Cys 130 18 133 PRT Rattus sp. 18 Gly Thr Leu Ile Glu Ser LeuGlu Ser Leu Lys Asn Tyr Phe Asn Ser 1 5 10 15 Ser Ser Met Asp Ala MetGlu Gly Lys Ser Leu Leu Leu Asp Ile Trp 20 25 30 Arg Asn Trp Gln Lys AspGly Asn Thr Lys Ile Leu Glu Ser Gln Ile 35 40 45 Ile Ser Phe Tyr Leu ArgLeu Phe Glu Val Leu Lys Asp Asn Gln Ala 50 55 60 Ile Ser Asn Asn Ile SerVal Ile Glu Ser His Leu Ile Thr Asn Phe 65 70 75 80 Phe Ser Asn Ser LysAla Lys Lys Asp Ala Phe Met Ser Ile Ala Lys 85 90 95 Phe Glu Val Asn AsnPro Gln Ile Gln His Lys Ala Val Asn Glu Leu 100 105 110 Ile Arg Val IleHis Gln Leu Ser Pro Glu Ser Ser Leu Arg Lys Arg 115 120 125 Lys Arg SerArg Cys 130 19 41 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 19 Xaa Leu Thr Asn Tyr Ser Val Thr Asp LeuAsn Val Gln Arg Lys Ala 1 5 10 15 Ile His Glu Leu Ile Gln Val Met AlaGlu Leu Ser Pro Ala Ala Lys 20 25 30 Thr Gly Lys Arg Lys Arg Ser Gln Met35 40 20 39 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 20 Xaa Ala Lys Phe Glu Val Asn Asn Pro Gln Val Gln ArgGln Ala Phe 1 5 10 15 Asn Glu Leu Ile Arg Val Val His Gln Leu Leu ProGlu Ser Ser Leu 20 25 30 Arg Lys Arg Lys Arg Ser Arg 35 21 21 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide21 Xaa Ile Arg Val Val His Gln Leu Leu Pro Glu Ser Ser Leu Arg Lys 1 510 15 Arg Lys Arg Ser Arg 20 22 38 PRT Mus sp. 22 Ala Lys Phe Glu ValAsn Asn Pro Gln Val Gln Arg Gln Ala Phe Asn 1 5 10 15 Glu Leu Ile ArgVal Val His Gln Leu Leu Pro Glu Ser Ser Leu Arg 20 25 30 Lys Arg Lys ArgSer Arg 35 23 38 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 23 Pro Ser Arg Glu Asn Gln Asn Ala Val LysIle Gln Lys Leu Ser Val 1 5 10 15 Val Leu Arg Arg Glu Gln Lys His ArgVal Glu Arg Leu Ala Phe Arg 20 25 30 Asn Gln Ser Leu Pro Phe 35 24 39PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 24 Xaa Pro Ser Arg Glu Asn Gln Asn Ala Val Lys Ile Gln Lys LeuSer 1 5 10 15 Val Val Leu Arg Arg Glu Gln Lys His Arg Val Glu Arg LeuAla Phe 20 25 30 Arg Asn Gln Ser Leu Pro Phe 35 25 12 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 25 Xaa GluSer Ser Leu Arg Lys Arg Lys Arg Ser Arg 1 5 10 26 9 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 26 Glu AspAla Glu Leu Asn Pro Arg Phe 1 5 27 9 PRT Artificial Sequence Descriptionof Artificial Sequence Synthetic peptide 27 Ser Glu Leu Asp Leu Glu LysGly Leu 1 5 28 9 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 28 Asp Glu Leu Glu Ala Val Pro Asn Ile 1 5 299 PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 29 Lys Glu Asp Ala Leu Gln Arg Pro Val 1 5 30 9 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 30 Glu AspAla Leu Gln Arg Pro Val Ala 1 5 31 9 PRT Artificial Sequence Descriptionof Artificial Sequence Synthetic peptide 31 Gly Glu Lys Leu Arg Val LeuGly Tyr 1 5 32 9 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 32 Glu Asp Thr Met Glu Val Glu Glu Phe 1 5 339 PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 33 Met Glu Tyr Leu Glu Lys Lys Asn Phe 1 5 34 9 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 34 Asn GluGlu Ala Ala Asp Glu Val Phe 1 5 35 9 PRT Artificial Sequence Descriptionof Artificial Sequence Synthetic peptide 35 Val Asn Gln Glu Arg Phe ArgMet Ile 1 5 36 9 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 36 Leu Phe Gln Lys Leu Ala Ser Gln Leu 1 5 379 PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 37 Ala Arg Lys Leu Arg His Val Phe Leu 1 5 38 9 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 38 Ala LeuLys Ile Lys Ile Ser Gln Ile 1 5 39 8 PRT Artificial Sequence Descriptionof Artificial Sequence Synthetic peptide 39 Cys Val Lys Leu Gln Thr ValHis 1 5 40 9 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 40 Lys Ala Leu Gln Arg Pro Val Ala Ser 1 5 41 9 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide41 Gly Ala Lys Thr Lys Ala Thr Ser Leu 1 5 42 9 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 42 Ile Gln Gln MetArg Asn Lys Phe Ala 1 5 43 8 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 43 Gln Met Arg Asn Lys Phe Ala Phe1 5 44 9 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 44 Asn Pro Arg Phe Leu Lys Asp Asn Leu 1 5 45 10 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide45 Thr Pro Asp Cys Ser Ser Asn Glu Asn Leu 1 5 10 46 9 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 46 Thr ProArg Arg Gln Ser Met Thr Val 1 5 47 9 PRT Artificial Sequence Descriptionof Artificial Sequence Synthetic peptide 47 Ser Pro Gly Gln Arg Ser IleSer Leu 1 5 48 10 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 48 His Pro Asn Leu Val Gln Leu Leu Gly Val 15 10 49 10 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 49 Ser Pro Lys Pro Ser Asn Gly Ala Gly Val 1 5 10 50 9PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 50 Lys Pro Leu Arg Arg Gln Val Thr Val 1 5 51 9 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 51 Ser ProAla Pro Val Pro Ser Thr Leu 1 5 52 9 PRT Artificial Sequence Descriptionof Artificial Sequence Synthetic peptide 52 Glu Arg Cys Lys Ala Ser IleArg Arg 1 5 53 10 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 53 Asp Arg Gln Arg Trp Gly Phe Phe Arg Arg 15 10 54 9 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 54 Gln Arg Val Gly Asp Leu Phe Gln Lys 1 5 55 9 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide55 Phe Arg Val His Ser Arg Asn Gly Lys 1 5 56 9 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 56 Leu Leu Tyr LysPro Val Asp Arg Val 1 5 57 9 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 57 Phe Leu Ser Ser Ile Asn Glu GluIle 1 5 58 9 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 58 Gln Leu Leu Lys Asp Ser Phe Met Val 1 5 59 9 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide59 Phe Met Val Glu Leu Val Glu Gly Ala 1 5 60 9 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 60 Lys Leu Ser GluGln Glu Ser Leu Leu 1 5 61 9 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 61 Met Leu Thr Asn Ser Cys Val LysLeu 1 5 62 10 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 62 Gly Leu Tyr Gly Phe Leu Asn Val Ile Val 1 5 10 63 9PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 63 Phe Leu Asn Val Thr Val His Ser Ala 1 5 64 9 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 64 Val LeuLeu Tyr Met Ala Thr Gln Ile 1 5 65 9 PRT Artificial Sequence Descriptionof Artificial Sequence Synthetic peptide 65 Phe Ile Pro Leu Ile Ser ThrArg Val 1 5 66 9 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 66 Val Val Leu Asp Ser Thr Glu Ala Leu 1 5 679 PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 67 Asn Gln Glu Arg Phe Arg Met Ile Tyr 1 5 68 9 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 68 Val GlyAsp Ala Ser Arg Pro Pro Tyr 1 5 69 10 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 69 Lys Val Pro GluLeu Tyr Glu Ile His Lys 1 5 10 70 9 PRT Artificial Sequence Descriptionof Artificial Sequence Synthetic peptide 70 Lys Leu Ala Ser Gln Leu GlyVal Tyr 1 5 71 10 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 71 Met Val Glu Leu Val Glu Gly Ala Arg Lys 15 10 72 9 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 72 Ile Val His Ser Ala Thr Gly Phe Lys 1 5 73 9 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide73 Ala Thr Gly Phe Lys Gln Ser Ser Lys 1 5 74 9 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 74 Lys Gln Ser SerLys Ala Leu Gln Arg 1 5 75 10 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 75 Glu Val Tyr Glu Gly Val Trp LysLys Tyr 1 5 10 76 10 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 76 Ser Leu Ala Tyr Asn Lys Phe Ser Ile Lys 15 10 77 9 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 77 Asn Leu Phe Ser Ala Leu Ile Lys Lys 1 5 78 11 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide78 Thr Val Ala Pro Ala Ser Gly Leu Pro His Lys 1 5 10 79 10 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide79 Leu Ile Ser Thr Arg Val Ser Leu Arg Lys 1 5 10 80 9 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 80 Arg IleAla Ser Gly Ala Ile Thr Lys 1 5 81 13 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 81 Lys Gln Ser SerLys Ala Leu Gln Arg Pro Val Ala Ser 1 5 10 82 11 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 82 Arg Val Glu GlyAsn Leu Ala Arg Val Glu Tyr 1 5 10 83 9 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 83 Gly Ser Asp CysThr Thr Ile His Tyr 1 5 84 11 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 84 Leu Leu Pro Glu Asn Asn Val LeuSer Pro Leu 1 5 10 85 9 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 85 Arg Met Pro Glu Ala Ala Pro ProVal 1 5 86 9 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 86 Arg Met Pro Glu Ala Ala Pro Arg Val 1 5 87 9 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide87 Ala Leu Asn Lys Met Phe Cys Gln Leu 1 5 88 9 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 88 Ser Thr Pro ProPro Gly Thr Arg Val 1 5 89 11 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 89 Gly Leu Ala Pro Pro Gln His LeuIle Arg Val 1 5 10 90 9 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 90 Leu Leu Gly Arg Asn Ser Phe GluVal 1 5 91 9 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 91 Pro Leu Asp Gly Glu Tyr Phe Thr Leu 1 5 92 9 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide92 Arg Val Arg Ala Met Ala Ile Tyr Lys 1 5 93 9 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 93 Arg Arg Thr GluGlu Glu Asn Leu Arg 1 5 94 9 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 94 Glu Leu Pro Pro Gly Ser Thr LysArg 1 5 95 10 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 95 Leu Pro Glu Asn Asn Val Leu Ser Pro Leu 1 5 10 9611 PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 96 Ala Pro Arg Met Pro Glu Ala Ala Pro Pro Val 1 5 10 97 11 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide97 Ala Pro Arg Met Pro Glu Ala Ala Pro Arg Val 1 5 10 98 9 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide98 Ala Pro Pro Gln His Leu Ile Arg Val 1 5 99 9 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 99 Arg Pro Ile LeuThr Ile Ile Thr Leu 1 5 100 11 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 100 Lys Pro Leu Asp Gly Glu ThrTyr Phe Thr Leu 1 5 10 101 9 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 101 Cys Gln Leu Ala Lys Thr CysPro Val 1 5 102 9 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 102 Gly Leu Ala Pro Pro Gln His Leu Ile 1 5103 9 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 103 Asn Thr Phe Arg His Ser Val Val Val 1 5 104 11 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide104 Leu Leu Pro Glu Asn Asn Val Leu Ser Pro Leu 1 5 10 105 9 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide105 Arg Met Pro Glu Ala Ala Pro Pro Val 1 5 106 10 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 106 LeuIle Arg Val Glu Gly Asn Leu Arg Val 1 5 10 107 9 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 107 Met Leu Leu AlaVal Leu Tyr Cys Leu 1 5 108 9 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 108 Tyr Met Asn Gly Thr Met SerGln Val 1 5 109 9 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 109 Tyr Met Asp Gly Thr Met Ser Gln Val 1 5110 9 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 110 Ala Phe Leu Pro Trp His Arg Leu Phe 1 5 111 9 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide111 Ser Glu Ile Trp Arg Asp Ile Asp Phe 1 5 112 15 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 112 GlnAsn Ile Leu Leu Ser Asn Ala Pro Leu Gly Pro Gln Phe Pro 1 5 10 15 113 13PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 113 Ser Tyr Leu Gln Asp Ser Asp Pro Asp Ser Phe Gln Asp 1 5 10114 9 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 114 Lys Thr Trp Gly Gln Tyr Trp Gln Val 1 5 115 10 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide115 Ala Met Leu Gly Thr His Thr Met Glu Val 1 5 10 116 9 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 116 MetLeu Gly Thr His Thr Met Glu Val 1 5 117 9 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 117 Ile Thr Asp GlnVal Pro Phe Ser Val 1 5 118 9 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 118 Tyr Leu Glu Pro Gly Pro ValThr Ala 1 5 119 10 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 119 Leu Leu Asp Gly Thr Ala Thr Leu Arg Leu 15 10 120 10 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 120 Val Leu Tyr Arg Tyr Gly Ser Phe Ser Val 1 5 10 12110 PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 121 Ser Leu Ala Asp Thr Asn Ser Leu Ala Val 1 5 10 122 9 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide122 Ala Leu Leu Ala Val Gly Ala Thr Lys 1 5 123 10 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 123 GluAla Ala Gly Ile Gly Ile Leu Thr Val 1 5 10 124 9 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 124 Ile Leu Thr ValIle Leu Gly Val Leu 1 5 125 9 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 125 Met Ser Leu Gln Arg Gln PheLeu Arg 1 5 126 9 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 126 Leu Leu Gly Pro Gly Arg Pro Tyr Arg 1 5127 9 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 127 Glu Glu Lys Leu Ile Val Val Leu Phe 1 5 128 10 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide128 Ala Cys Asp Pro His Ser Gly His Phe Val 1 5 10 129 9 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 129 SerTyr Leu Asp Ser Gly Ile His Phe 1 5 130 9 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 130 Phe Pro Ser AspSer Trp Cys Tyr Phe 1 5 131 9 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 131 Glu Ala Asp Pro Thr Gly HisSer Tyr 1 5 132 9 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 132 Ser Ala Tyr Gly Glu Pro Arg Lys Leu 1 5133 9 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 133 Glu Val Asp Pro Ile Gly His Leu Tyr 1 5 134 9 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide134 Phe Leu Trp Gly Pro Arg Ala Leu Val 1 5 135 10 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 135 MetGlu Val Asp Pro Ile Gly His Leu Tyr 1 5 10 136 9 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 136 Ala Ala Arg AlaVal Phe Leu Ala Leu 1 5 137 8 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 137 Tyr Arg Pro Arg Pro Arg ArgTyr 1 5 138 10 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 138 Ser Pro Ser Ser Asn Arg Ile Arg Asn Thr 15 10 139 9 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 139 Val Leu Pro Asp Val Phe Ile Arg Cys 1 5 140 20 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide140 Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly 1 510 15 Val Thr Ser Ala 20 141 11 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 141 Gly Leu Glu Gly Leu Ile HisSer Gln Arg Arg 1 5 10 142 8 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 142 Phe Pro Asp Trp Gln Asn TyrThr 1 5 143 9 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 143 Leu Thr Phe Gly Trp Cys Tyr Lys Leu 1 5 144 8 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide144 Arg Phe Asp Ser Arg Leu Ala Phe 1 5 145 8 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 145 Ala Arg Glu LeuHis Pro Glu Tyr 1 5 146 9 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 146 Lys Leu Thr Pro Leu Cys ValThr Leu 1 5 147 9 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 147 Ser Leu Tyr Asn Thr Val Ala Thr Leu 1 5148 9 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 148 Ala Leu Val Glu Ile Cys Thr Glu Met 1 5 149 11 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide149 Val Leu Asp Val Gly Asp Ala Tyr Phe Ser Val 1 5 10 150 9 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide150 Lys Ile Tyr Gln Tyr Met Asp Asp Leu 1 5 151 9 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 151 LysIle Glu Glu Leu Arg Gln His Leu 1 5 152 11 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 152 Leu Leu Arg TrpGly Leu Thr Thr Pro Asp Lys 1 5 10 153 9 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 153 Ile Leu Lys GluPro Val His Gly Val 1 5 154 9 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 154 Pro Leu Val Lys Leu Trp TyrGln Leu 1 5 155 9 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 155 Glu Leu Val Asn Gln Asp Glu Gln Leu 1 5156 10 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 156 Pro Leu Thr Phe Gly Trp Cys Phe Lys Leu 1 5 10 15710 PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 157 Val Leu Gln Trp Arg Phe Asp Ser Arg Leu 1 5 10 158 9 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide158 Ala Leu His His Val Ala Arg Glu Leu 1 5 159 9 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 159 SerLeu Leu Asn Ala Thr Val Asp Ile 1 5 160 9 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 160 Asp Leu Asn ThrMet Leu Asn Thr Val 1 5 161 9 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 161 Val Ile Tyr Gln Tyr Met AspAsp Leu 1 5 162 9 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 162 Pro Leu Val Lys Leu Trp Tyr Gln Leu 1 5163 10 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 163 Val Leu Val Gly Pro Thr Pro Val Asn Ile 1 5 10 1648 PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 164 Thr Val Tyr Tyr Gly Val Pro Val 1 5 165 8 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 165 SerLeu Lys Pro Cys Val Lys Leu 1 5 166 9 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 166 Arg Ile Gln ArgGly Pro Gly Arg Ala 1 5 167 9 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 167 Thr Leu Thr Ser Cys Asn ThrSer Val 1 5 168 8 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 168 Phe Ile Asn Met Trp Gln Glu Val 1 5 169 9PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 169 Val Val Gln Gly Ala Tyr Arg Ala Ile 1 5 170 10 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide170 His Ala Gly Pro Ile Ala Pro Gly Gln Met 1 5 10 171 10 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 171 GlnMet Lys Asp Cys Thr Glu Arg Gln Ala 1 5 10 172 9 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 172 Phe Leu Gln SerArg Pro Glu Thr Ala 1 5 173 11 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 173 Glu Ser Glu Leu Val Asn GlnIle Ile Glu Gly 1 5 10 174 9 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 174 Lys Ile Arg Leu Arg Pro GlyGly Lys 1 5 175 9 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 175 Arg Leu Arg Pro Gly Gly Lys Lys Lys 1 5176 11 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 176 Ala Leu Val Glu Ile Cys Thr Glu Met Glu Lys 1 5 10177 9 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 177 Ala Ile Phe Gln Ser Ser Met Thr Lys 1 5 178 10 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide178 Asp Leu Glu Ile Gly Gln His Arg Thr Lys 1 5 10 179 10 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 179 GlnVal Pro Leu Arg Pro Met Thr Tyr Lys 1 5 10 180 10 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 180 ThrVal Tyr Tyr Gly Val Pro Val Trp Lys 1 5 10 181 11 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 181 ArgLeu Arg Asp Leu Leu Leu Ile Val Thr Arg 1 5 10 182 9 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 182 LysIle Arg Leu Arg Pro Gly Gly Lys 1 5 183 9 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 183 Ala Ile Phe GlnSer Ser Met Thr Lys 1 5 184 11 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 184 Gln Ile Tyr Gln Glu Pro PheLys Asn Leu Lys 1 5 10 185 10 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 185 Gln Val Pro Leu Arg Pro MetThr Tyr Lys 1 5 10 186 9 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 186 Ala Val Asp Leu Ser His PheLeu Lys 1 5 187 10 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 187 Ala Cys Gln Val Gly Gly Pro Gly His Lys 15 10 188 9 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 188 Ala Thr Leu Tyr Cys Val His Gln Arg 1 5 189 10 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide189 Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr 1 5 10 190 8 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 190 TyrLeu Lys Asp Gln Gln Leu Leu 1 5 191 9 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 191 Arg Tyr Leu LysAsp Gln Gln Leu Leu 1 5 192 9 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 192 Val Tyr Tyr Asp Pro Ser LysAsp Leu 1 5 193 9 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 193 Ile Tyr Gln Glu Pro Phe Lys Asn Leu 1 5194 11 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 194 Glu Ser Glu Leu Val Asn Gln Ile Ile Glu Gly 1 5 10195 10 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 195 Glu Thr Ile Asn Glu Glu Ala Ala Glu Trp 1 5 10 1969 PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 196 Glu Val Ile Pro Met Phe Ser Ala Leu 1 5 197 9 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 197 ArgAla Ile Arg His Ile Pro Arg Arg 1 5 198 11 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 198 Arg Leu Arg AspLeu Leu Leu Ile Val Thr Arg 1 5 10 199 9 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 199 Arg Ile Lys GlnIle Ile Asn Met Trp 1 5 200 9 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 200 His Arg Leu Arg Asp Leu LeuLeu Ile 1 5 201 10 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 201 Pro Ile Gln Lys Glu Thr Trp Glu Thr Trp 15 10 202 10 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 202 Ile Ile Leu Gly Leu Asn Lys Ile Val Arg 1 5 10 2039 PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 203 Tyr Leu Ala Trp Val Pro Ala His Lys 1 5 204 9 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 204 PhePro Val Thr Gln Val Pro Leu Arg 1 5 205 10 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 205 Thr Pro Gly ProGly Val Arg Tyr Pro Leu 1 5 10 206 10 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 206 Arg Pro Asn AsnAsn Thr Arg Lys Ser Ile 1 5 10 207 9 PRT Artificial Sequence Descriptionof Artificial Sequence Synthetic peptide 207 Ile Pro Arg Arg Ile Arg GlnGly Leu 1 5 208 9 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 208 Tyr Leu Ala Trp Val Pro Ala His Lys 1 5209 9 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 209 Arg Val Lys Glu Lys Tyr Gln His Leu 1 5 210 9 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide210 Gly Gly Lys Lys Lys Tyr Lys Leu Lys 1 5 211 8 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 211 PheLeu Lys Glu Lys Gly Gly Leu 1 5 212 9 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 212 Gly Glu Ile TyrLys Arg Trp Ile Ile 1 5 213 8 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 213 Tyr Leu Lys Asp Gln Gln LeuLeu 1 5 214 8 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 214 Pro Arg Arg Ile Arg Gln Gly Leu 1 5 215 10 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide215 Arg Ile Arg Gln Gly Leu Glu Arg Ile Leu 1 5 10 216 9 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 216 AspCys Lys Thr Ile Leu Lys Ala Leu 1 5 217 9 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 217 Gly Pro Lys ValLys Gln Trp Pro Leu 1 5 218 9 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 218 Glu Trp Arg Phe Asp Asp SerArg Leu 1 5 219 9 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 219 Glu Arg Tyr Leu Lys Asp Gln Gln Leu 1 5220 9 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 220 Asp Arg Phe Tyr Lys Thr Leu Arg Ala 1 5 221 9 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide221 Asp Leu Asn Thr Met Leu Asn Thr Val 1 5 222 11 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 222 LeuArg Ala Glu Gln Ala Ser Val Gln Glu Val 1 5 10 223 10 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 223 ArgAla Glu Gln Ala Ser Val Gln Glu Val 1 5 10 224 9 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 224 Tyr Pro Leu ThrPhe Gly Trp Cys Tyr 1 5 225 9 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 225 Tyr Pro Leu Thr Phe Gly TrpCys Phe 1 5 226 10 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 226 Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys 15 10 227 10 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 227 Gln Val Pro Leu Arg Pro Met Thr Tyr Lys 1 5 10 2288 PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 228 Arg Tyr Pro Leu Thr Phe Gly Trp 1 5 229 7 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 229 TyrPro Leu Thr Phe Gly Trp 1 5 230 9 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 230 Glu Arg Tyr Leu Lys Asp GlnGln Leu 1 5 231 10 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 231 Gly Arg Arg Gly Trp Glu Ala Leu Lys Tyr 15 10 232 9 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 232 Asp Pro Asn Pro Gln Glu Val Val Leu 1 5 233 9 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide233 Arg Pro Val Val Ser Thr Gln Leu Leu 1 5 234 10 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 234 ValPro Leu Asp Lys Asp Phe Arg Lys Tyr 1 5 10 235 9 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 235 Ser Pro Ala IlePhe Gln Ser Ser Met 1 5 236 9 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 236 Asn Pro Asp Ile Val Ile TyrGln Tyr 1 5 237 9 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 237 Ile Pro Leu Thr Glu Glu Ala Glu Leu 1 5238 10 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 238 Glu Pro Ile Val Gly Ala Glu Thr Phe Tyr 1 5 10 2399 PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 239 Phe Pro Val Arg Pro Gln Val Pro Leu 1 5 240 8 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 240 ValPro Leu Arg Pro Met Thr Tyr 1 5 241 9 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 241 Thr Ala Val ProTrp Asn Ala Ser Trp 1 5 242 11 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 242 Val Pro Val Trp Lys Glu AlaThr Thr Thr Leu 1 5 10 243 9 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 243 Asn Ser Ser Gln Val Ser GlnAsn Tyr 1 5 244 9 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 244 Pro Pro Ile Pro Val Gly Glu Ile Tyr 1 5245 9 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 245 Tyr Phe Pro Asp Trp Gln Asn Tyr Thr 1 5 246 9 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide246 Ser Glu Gly Ala Thr Pro Gln Asp Leu 1 5 247 10 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 247 LeuGlu Ser Gly Ala Thr Pro Gln Asp Leu 1 5 10 248 9 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 248 Arg Ala Ile GluAla Gln Gln His Leu 1 5 249 9 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 249 Ala Leu Val Glu Ile Cys ThrGlu Met 1 5 250 9 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 250 Glu Lys Glu Gly Lys Ile Ser Lys Ile 1 5251 8 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 251 Thr Ala Phe Thr Ile Pro Ser Ile 1 5 252 9 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide252 Ala Phe His His Val Ala Arg Glu Leu 1 5 253 11 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 253 ValPro Val Trp Lys Glu Ala Thr Thr Thr Leu 1 5 10 254 10 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 254 ThrSer Leu Thr Gln Glu Gln Ile Gly Trp 1 5 10 255 10 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 255 HisThr Gln Gly Tyr Phe Pro Asp Trp Gln 1 5 10 256 9 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 256 His Thr Gln GlyTyr Phe Pro Asp Trp 1 5 257 7 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 257 Tyr Phe Pro Asp Trp Gln Asn 15 258 9 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 258 Ile Ser Pro Arg Thr Leu Asn Ala Trp 1 5 259 9 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide259 Phe Ser Pro Glu Val Ile Pro Met Phe 1 5 260 10 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 260 ArgLeu Arg Pro Gly Gly Lys Lys Lys Tyr 1 5 10 261 10 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 261 LeuGly Leu Asn Lys Ile Val Arg Met Tyr 1 5 10 262 12 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 262 LeuVal Gly Lys Leu Asn Trp Ala Ser Gln Ile Tyr 1 5 10 263 8 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 263 AlaVal Asp Leu Ser His Phe Leu 1 5 264 11 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 264 Thr Gln Gly TyrPhe Pro Asp Trp Gln Asn Tyr 1 5 10 265 9 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 265 Ser Phe Asn CysGly Gly Glu Phe Phe 1 5 266 10 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 266 Val Thr Asp Ser Gln Tyr AlaLeu Gly Ile 1 5 10 267 9 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 267 Arg Ala Glu Gln Ala Ser GlnGlu Val 1 5 268 10 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 268 Lys Ala Ala Leu Asp Leu Ser His Pro Leu 15 10 269 9 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 269 Gln Ala Thr Gln Glu Val Lys Asn Trp 1 5 270 10 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide270 Tyr Met Leu Asp Leu Gln Pro Glu Thr Thr 1 5 10 271 9 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 271 LeuLeu Met Gly Thr Leu Gly Ile Val 1 5 272 8 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 272 Thr Leu Gly IleVal Cys Pro Ile 1 5 273 10 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 273 Thr Ile His Asp Ile Ile LeuGlu Cys Val 1 5 10 274 9 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 274 Lys Leu Pro Gln Leu Cys ThrGlu Leu 1 5 275 8 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 275 Arg Pro Pro Lys Leu Pro Gln Leu 1 5 27623 PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 276 Leu Arg Arg Glu Val Tyr Asp Phe Ala Phe Arg Asp Leu Cys IleVal 1 5 10 15 Tyr Arg Asp Gly Asn Pro Tyr 20 277 9 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 277 IleSer Glu Tyr Arg His Tyr Cys Tyr 1 5 278 20 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 278 Glu Lys Gln ArgHis Leu Asp Lys Lys Gln Arg Phe His Asn Ile Arg 1 5 10 15 Gly Arg TrpThr 20 279 15 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 279 Gly Gln Ala Glu Pro Asp Arg Ala His Tyr Asn IleVal Thr Phe 1 5 10 15 280 9 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 280 Gln Ala Glu Pro Asp Arg AlaHis Tyr 1 5 281 10 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 281 Glu Pro Asp Arg Ala His Tyr Asn Ile Val 15 10

What is claimed is:
 1. An isolated lipopeptide, comprising: a peptidewhich binds to an interferon-gamma (IFN-γ) receptor intracellularly, butwhich does not bind to the IFN-γ receptor extracellularly, said peptideconsists of a peptide sequence delimited by amino acids located inpositions 95 and 133 or 132 of murine LFN-γ, reference SEQ ID NO:17; anda lipophilic molecule selected from the group consisting of a linear orbranched, saturated or unsaturated C4 to C20 hydrocarbon-based chain,and a steroid group, said steroid group optionally linked to saidhydrocarbon-based chain, wherein said lipophilic molecule is covalentlylinked to the peptide and is optionally combined with a short vectorpeptide comprising a group that is ionized at physiological pH, and agroup for covalently bonding to said hydrocarbon-based chain or saidsteroid group.
 2. The lipopeptide according to claim 1, wherein saidlipophilic molecule is a hydrocarbon-based chain selected from the groupconsisting of palmitic acid, oleic acid, linoleic acid, and linolenicacid.
 3. The lipopeptide according to claim 1, wherein said lipophilicmolecule is a steroid group, said steroid group is a cholesterolselected from the group consisting of cholest-5-enyl-3-oxyacetic acidand cholest-5-enyl-3-oxycarbonic acid.
 4. The lipopeptide according toclaim 1, wherein said lipophilic molecule is covalently bonded to one ormore amino acids of the peptide.
 5. The lipopeptide according to claim1, wherein said lipophilic molecule is covalently bonded to an αNH₂ orεNH₂ group of a lysine located in N-terminal or C-terminal position ofthe peptide, or to any amino, alcohol or thiol group optionally added tothe peptide with a spacer.
 6. The lipopeptide according to claim 1,wherein a COOH group of the C-terminal amino acid is substituted with agroup and the substituted lipopentide is resistant to exopeptidases. 7.The lipopeptide according to claim 1, wherein the lipophilic molecule isN^(α)-acetyl-Lysine N^(ε)(palmitoyl) group, and wherein the lipopeptideis selected from the group consisting of a sequence delimited by theamino acids located in positions 95 and 132 of the murine IFN-γ peptidesequence (reference SEQ ID NO:17), consisting of SEQ ID NO:20, and asequence delimited by the amino acids located in positions 113 and 132of the murine IFN-γ peptide sequence, consisting of SEQ ID NO:21.
 8. Amicelle or micro-aggregate of the lipopeptide according to claim 1,wherein said micelle or micro-aggregate is less than 1 μm in size. 9.The micelle or micro-aggregate according to claim 8, obtained bydispersing the lipopeptide in about 80% concentrated acetic acidsolution.
 10. A pharmaceutical composition comprising a lipopeptideaccording to claim 1, in combination with a carrier in a physiologicallyacceptable pharmaceutical formulation.
 11. An isolated peptide delimitedon the N-terminal side by an amino acid located in position 113 to 121,and on the C-terminal side by an amino acid located in position 132 ofthe murine IFN-γ peptide sequence (reference SEQ ID NO:17), representedby SEQ ID NO:1.